Supporting information for. Determination of sub nm levels of low molecular mass (LMM) thiols in natural waters by liquid

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1 Supporting information for Determination of sub nm levels of low molecular mass (LMM) thiols in natural waters by liquid chromatography tandem mass spectrometry after derivatization with p-(hydroxymercuri) benzoate and online pre-concentration Van Liem-Nguyen, Sylvain Bouchet,, Erik Björn*, Department of Chemistry, Umeå University, SE Umeå, Sweden Present address: IPREM-LCABIE, CNRS UMR 5254, Université de Pau et des Pays de l'adour, Technopole Helioparc, 2, avenue du Président Angot PAU cedex 09 *Corresponding author 1

2 Table of Contents Figure S-1. Structures and abbreviations used for the 16 thiols selected for this study Table S-1. Mobile phase compositions and flow rates used to load the analytes on the SPE cartridge and then washing and stabilizing it. After the 2 min loading step, the analytes are eluted from the SPE by a mobile phase gradient described below (Table S-2) Table S-2. Mobile phase compositions and flow rates used to elute the analytes out of the analytical column (Agilent Zorbax SB-C8 (2.1 x 100 mm, 3.5 µm) fitted with an Ultra C8 guard column (10 x 2.1 mm, 5 µm), according to the use of a SPE pre-concentration column or not Figure S-2. Zoom in the full scan spectra of 2-MPA (a), SUC (b), SULF (c), NACCys (d), Glyc (e), ETH (f) and Cyst (g) acquired in negative electrospray ionization by direct infusion of each thiol-phmb complex in 0.1%FA H2O/MeOH (50/50). The m/z value of the PHMB-thiol complex precursor ions (monoisotopic) are indicated in bold font Figure S-3. Zoom in the full scan spectra of 2-MPA (a), SUC (b), SULF (c), NACCys (d), Glyc (e), ETH (f) and Cyst (g) acquired in positive electrospray ionization by direct infusion of each thiol-phmb complex in 0.1%FA H2O/MeOH (50/50). The m/z value of the PHMB-thiol complex precursor ions (monoisotopic) are indicated in bold font Figure S-4. Optimization of the collision energies for the dissociation of 2-MPA (a), SUC (b), SULF (c), NACCys (d), Glyc (e), ETH (f) in negative, and Cyst (g) in positive electrospray ionization Figure S-5. High resolution ( ) mass spectra (Thermo LTQ Orbitrap) for the Cys (a) and SUC (b) complexes with PHMB and their fragments, allowing the identification of the daughter ion 355 m/z Figure S-6. High resolution mass spectra (60 000) (Thermo LTQ Orbitrap) for identification of common product ions in negative ionization (a) the produce 323 m/z of Glyc, (b) the produce 143 m/z of GSH, (c) the produce of 121 m/z of MAC, and in positive ionization (d) the produce of 383 m/z of Cyst Figure S-7. Recoveries of the 16 thiol-phmb complexes (300 nm each) by SPE pre-concentration in Mili-Q water at different ph; 24µM of each TCEP and PHMB, respectively. The peak areas were normalized to the ones obtained at ph 2.5 for each thiol (a) the normalized peak area of individual thiol-phmb, (b) the average normalized peak area of 16 thiol-phmb complexes (± 0.05, CI) Figure S-8. Recoveries of the 16 thiol-phmb complexes (300 nm each) added to a natural water sample contain 20 mg L -1 DOC by SPE pre-concentration at different ph, 24 µm of each TCEP and PHMB, respectively. The peak areas were normalized to the ones obtained at ph 2.5 for each thiol, 2

3 (a) the normalized peak area of individual thiol-phmb, (b) the average normalized peak area of 16 thiol-phmb complexes (± 0.05, CI) Figure S-9. The optimization of the PHMB to TCEP ratio for derivatization of thiol standards in Milli-Q water with SPE pre-concentration. The concentration of TCEP and total thiols were kept constant at 24 µm and 4.8 µm while varying the concentration of PHMB (ph 3.5). The peak areas were normalized to the ones obtained at the PHMB/TCEP ratio of 0.4 for each thiol, (a) the normalized peak area of individual thiol-phmb, (b) the average normalized peak area of 16 thiol-phmb complexes (± 0.05, CI) Figure S-10. The optimization of the PHMB to TCEP ratios for derivatization of thiol standards in natural water sample containing 20 mg L -1 DOC with SPE pre-concentration. The concentration of TCEP and total thiols were kept constant at 24 µm and 4.8 µm while varying the concentration of PHMB, (ph 3.5). The peak areas were normalized to the ones obtained at the PHMB/TCEP ratio of 0.4 for each thiol, (a) the normalized peak area of individual thiol-phmb, (b) the average normalized peak area of 16 thiol-phmb complexes (± 0.05, CI) Figure S-11. Effect of the PHMB to thiol ratio on the thiol-phmb complexes peak area (300 nm each) in Milli-Q water with SPE pre-concentration (TCEP/PHMB =1, ph 3.5). The peak areas were normalized to the ones obtained at the PHMB/thiol ratio of 1 for each thiol, (a) the normalized peak area of individual thiol-phmb, (b) the average normalized peak area of 16 thiol-phmb complexes (± 0.05, CI) Figure S-12. Effect of the PHMB to thiol ratio on the thiol-phmb complexes peak area (300 nm each) in a natural water contain 20 mg L -1 DOC with SPE pre-concentration (TCEP/PHMB =1, ph 3.5). The peak areas were normalized to the ones obtained at the PHMB/thio ratio of 1 for each thiol, (a) the normalized peak area of individual thiol-phmb, (b) the average normalized peak area of 16 thiol- PHMB complexes (± 0.05, CI) Figure S-13. Normalized peak area of thiol standards at different free sulfide concentrations. The total thiol concentration was 4.8 µm and concentrations of PHMB and TCEP were 24 µm, ph 3.5. The peak areas were normalized to the ones obtained without addition of sulfide, (a) the normalized peak area of individual thiol-phmb, (b) the average normalized peak area of 16 thiol-phmb complexes (± 0.05, CI) Table S-3. The SPE recoveries of each thiol-phmb complex under various matrix conditions Figure S-14. Calibration curves of 16 thiols obtained without SPE pre-concentration (ph 3.3) Figure S-15. Calibration curves of 16 thiols obtained with 1 ml SPE pre-concentration (ph 3.3)

4 Figure S-16: Stability over time of thiol-phmb complexes (300 nm of each thiol) in a natural water sample containing 20 mg L -1 DOC for different storage temperature (+4 o C and -20 o C) and different containers polypropylene (PP) and FEP Teflon (FT). Samples were analyzed using the SPE preconcentration procedure. The instrumental drift was corrected by running a set of freshly prepared thiol-phmb complexes Figure S-17. Precursor ion scan of a natural water sample containing 20 mg L-1 DOC and spiked with 1 µm of 3-mercaptopropansulfonate (3-Mer) and 5 µm of thiosalicylic acid (Thio). (a) relative abundance of the m/z 355 target ion and retention time of the corresponding precursor compounds, the precursor ion mass spectrum (scanned from 300 to 1500 m/z in negative electrospray ionization) of (b) 3-Mer-PHMB at 11.9 min, (c) Thio-PHMB at 20.9 min Figure S-18. Precursor ions scan of a mixture of 1 µm of Cys, Pen, NACCys and NACPen complexes with PHMB

5 Figure S-1. Structures and abbreviations used for the 16 thiols selected for this study. 5

6 Table S-1. Mobile phase compositions and flow rates used to load the analytes on the SPE cartridge and then washing and stabilizing it. After the 2 min loading step, the analytes are eluted from the SPE by a mobile phase gradient described below (Table S-2). Loading Washing loop Washing loop and SPE Stablizing SPE Time H 2 O MeOH Flow rate (min) 0.1%FA 0.1%FA ml min Table S-2. Mobile phase compositions and flow rates used to elute the analytes out of the analytical column (Agilent Zorbax SB-C8 (2.1 x 100 mm, 3.5 µm) fitted with an Ultra C8 guard column (10 x 2.1 mm, 5 µm), according to the use of a SPE pre-concentration column or not. Without SPE With SPE Time Time H 2 O MeOH Flow rate H 2 O MeOH Flow rate (min) (min) 0.1%FA 0.1%FA ml min %FA 0.1%FA ml min

7 Figure S-2. Zoom in the full scan spectra of 2-MPA (a), SUC (b), SULF (c), NACCys (d), Glyc (e), ETH (f) and Cyst (g) acquired in negative electrospray ionization by direct infusion of each thiol-phmb complex in 0.1%FA H2O/MeOH (50/50). The m/z value of the PHMB-thiol complex precursor ions (monoisotopic) are indicated in bold font. 7

8 Figure S-3. Zoom in the full scan spectra of 2-MPA (a), SUC (b), SULF (c), NACCys (d), Glyc (e), ETH (f) and Cyst (g) acquired in positive electrospray ionization by direct infusion of each thiol-phmb complex in 0.1%FA H2O/MeOH (50/50). The m/z value of the PHMB-thiol complex precursor ions (monoisotopic) are indicated in bold font. 8

9 Figure S-4. Optimization of the collision energies for the dissociation of 2-MPA (a), SUC (b), SULF (c), NACCys (d), Glyc (e), ETH (f) in negative, and Cyst (g) in positive electrospray ionization. 9

10 Figure S-5. High resolution ( ) mass spectra (Thermo LTQ Orbitrap) for the Cys (a) and SUC (b) complexes with PHMB and their fragments, allowing the identification of the daughter ion 355 m/z. 10

11 Relative Abundance Relative Abundance Relative Abundance Relative Abundance HO-C Hg O = = = = = Hg Relative Abundance Relative Abundance Hg Relative Abundance Relative Abundance Hg = = Figure S-6. High resolution mass spectra (60 000) (Thermo LTQ Orbitrap) for identification of common product ions in negative ionization (a) the produce 323 m/z of Glyc, (b) the produce 143 m/z of GSH, (c) the produce of 121 m/z of MAC, and in positive ionization (d) the produce of 383 m/z of Cyst. 11

12 Figure S-7. Recoveries of the 16 thiol-phmb complexes (300 nm each) by SPE pre-concentration in Mili-Q water at different ph; 24µM of each TCEP and PHMB, respectively. The peak areas were normalized to the ones obtained at ph 2.5 for each thiol (a) the normalized peak area of individual thiol-phmb, (b) the average normalized peak area of 16 thiol-phmb complexes (± 0.05, CI). 12

13 Figure S-8. Recoveries of the 16 thiol-phmb complexes (300 nm each) added to a natural water sample contain 20 mg L -1 DOC by SPE pre-concentration at different ph, 24 µm of each TCEP and PHMB, respectively. The peak areas were normalized to the ones obtained at ph 2.5 for each thiol, (a) the normalized peak area of individual thiol-phmb, (b) the average normalized peak area of 16 thiol-phmb complexes (± 0.05, CI). 13

14 Figure S-9. The optimization of the PHMB to TCEP ratio for derivatization of thiol standards in Milli-Q water with SPE pre-concentration. The concentration of TCEP and total thiols were kept constant at 24 µm and 4.8 µm while varying the concentration of PHMB (ph 3.5). The peak areas were normalized to the ones obtained at the PHMB/TCEP ratio of 0.4 for each thiol, (a) the normalized peak area of individual thiol-phmb, (b) the average normalized peak area of 16 thiol-phmb complexes (± 0.05, CI). 14

15 Figure S-10. The optimization of the PHMB to TCEP ratios for derivatization of thiol standards in natural water sample containing 20 mg L -1 DOC with SPE pre-concentration. The concentration of TCEP and total thiols were kept constant at 24 µm and 4.8 µm while varying the concentration of PHMB, (ph 3.5). The peak areas were normalized to the ones obtained at the PHMB/TCEP ratio of 0.4 for each thiol, (a) the normalized peak area of individual thiol-phmb, (b) the average normalized peak area of 16 thiol-phmb complexes (± 0.05, CI). 15

16 Figure S-11. Effect of the PHMB to thiol ratio on the thiol-phmb complexes peak area (300 nm each) in Milli-Q water with SPE pre-concentration (TCEP/PHMB =1, ph 3.5). The peak areas were normalized to the ones obtained at the PHMB/thiol ratio of 1 for each thiol, (a) the normalized peak area of individual thiol-phmb, (b) the average normalized peak area of 16 thiol-phmb complexes (± 0.05, CI). 16

17 Figure S-12. Effect of the PHMB to thiol ratio on the thiol-phmb complexes peak area (300 nm each) in a natural water contain 20 mg L -1 DOC with SPE pre-concentration (TCEP/PHMB =1, ph 3.5). The peak areas were normalized to the ones obtained at the PHMB/thio ratio of 1 for each thiol, (a) the normalized peak area of individual thiol-phmb, (b) the average normalized peak area of 16 thiol- PHMB complexes (± 0.05, CI). 17

18 Figure S-13. Normalized peak area of thiol standards at different free sulfide concentrations. The total thiol concentration was 4.8 µm and concentrations of PHMB and TCEP were 24 µm, ph 3.5. The peak areas were normalized to the ones obtained without addition of sulfide, (a) the normalized peak area of individual thiol-phmb, (b) the average normalized peak area of 16 thiol-phmb complexes (± 0.05, CI). 18

19 Table S-3. The SPE recoveries of each thiol-phmb complex under various matrix conditions. Thiols 20 mg L -1 certified NOM 80 mg L -1 certified NOM 20 mg L -1 DOC fresh water -1 (2*) 20 mg L DOC fresh water 40 mg L -1 DOC fresh water ETH 67 ±5 73 ± 7-83 ± ± ± 6 MAC 65 ±4 75 ± 5-74 ± 9 92 ± 7 54 ± 10 2-MPA 69 ± ± ± ± 4 98 ± 9 71 ± 16 3-MPA 72 ± 3 96 ± 6 33 ±9 93 ± 4 79 ± ± 13 Glyc 76 ± ± ± ± 9 56 ± 13 Cys 80 ± 4 74 ± 6 21 ± 3 77 ± 5 77 ± 8 44 ± 8 Hcys 68 ± 4 78 ± 2 19 ± 4 71 ± 5 87 ± 7 45 ± 6 SULF 72 ± 6 81 ± 8 57 ± ± ± 9 84 ± 6 Pen 88 ± 3 72 ± 5 33 ± 9 95 ± ± 4 73 ± 6 SUC 68 ± 4 74 ± 3 85 ± ± ± 7 49 ± 20 NACCys 80 ± 3 93 ± 8 59 ± ± 8 78 ± 5 65 ± 14 CysGly 79 ± 3 79 ± 5 36 ±8 81 ± 3 76 ± ± 14 NACPen 68 ± ± 3 83 ± ± ± 5 86 ± 12 GluCys 71 ± 4 86± 5 53 ± 7 92 ± 4 83 ± 3 59 ± 15 GSH 86 ± 2 82 ± 8 28 ± 2 86 ± 4 79 ± 4 68 ± 5 Cyst 78 ± 3 74 ± 6 18 ± 2 96 ± 9 81 ± 6 77 ± 13 Average SD Not found SPE recovery for Milli-Q water matrix (1) (%) SPE recovery for NOM/DOC matrices relative to Milli-Q water (2) (%) (1) The recovery of each thiol-phmb complex in Milli-Q water using SPE pre-concentration (V inj = 1 ml at 300 nm each) compared to directly injected to analytical column (V inj = 10 µl at 30 µm each). For sample with SPE, ph of sample was adjusted to 3.1 while without SPE ph of sample was not measured. (2) The influence of certified NOM (included humic and fulvic material) and natural DOC concentrations on the recovery of complexes by the SPE cartridge (1 ml, ph 3.3). Each thiol was added at 300 nm, 33 µm of TCEP and 26 µm of PHMB were added for reduction and derivatization. (2*) The influence of natural DOC concentrations on the recovery of complexes by the SPE cartridge (1 ml, ph 3.3). Each thiol was added at 30 nm, 2.4 µm of TCEP and 2.4 µm of PHMB were added for reduction and derivatization. 19

20 Peak area Peak area y = (1.3 x + 0.4)ₓ10 5 R² = ₓ10 6 ₓ10 6 µm y = (7.2 x + 1.4)ₓ10 5 R² = y = (6.7 x + 2.8)ₓ10 5 R² = y = (3.6 x 3.5)ₓ10 5 R² = y = (2.9 x + 1.7)ₓ10 5 R² = y = (2.3 x -1.1)ₓ10 6 R² = y = (2.0 x + 0.1)ₓ10 6 R² = y = (1.3 x 1.1)ₓ10 6 R² = y = (8.8 x 8.2)ₓ10 5 R² = ETH MAC 2MPA 3MPA CysGly SULF Pen NACPen GluCys ₓ10 7 y = (2.6 x -1.3)ₓ10 6 ₓ10 8 R² = y = (2.1 x -1.4)ₓ10 6 R² = y = (14 x -0.2)ₓ10 5 R² = y = (4.9 x +5.5)ₓ10 5 R² = y = (4.8 x -4.0)ₓ10 5 R² = y = (2.9 x 2.0)ₓ10 7 R² = y = (2.7 x 2.0)ₓ10 5 R² = µm Cys Hcys SUC NACCys Glyc GSH Cyst Figure S-14. Calibration curves of 16 thiols obtained without SPE pre-concentration (ph 3.3). Peak area Peak area Figure S-15. Calibration curves of 16 thiols obtained with 1 ml SPE pre-concentration (ph 3.3). 20

21 Figure S-16: Stability over time of thiol-phmb complexes (300 nm of each thiol) in a natural water sample containing 20 mg L -1 DOC for different storage temperature (+4 o C and -20 o C) and different containers polypropylene (PP) and FEP Teflon (FT). Samples were analyzed using the SPE preconcentration procedure. The instrumental drift was corrected by running a set of freshly prepared thiol-phmb complexes. 21

22 22

23 Figure S-17. Precursor ion scan of a natural water sample containing 20 mg L-1 DOC and spiked with 1 µm of 3-mercaptopropansulfonate (3-Mer) and 5 µm of thiosalicylic acid (Thio). (a) relative abundance of the m/z 355 target ion and retention time of the corresponding precursor compounds, the precursor ion mass spectrum (scanned from 300 to 1500 m/z in negative electrospray ionization) of (b) 3-Mer-PHMB at 11.9 min, (c) Thio-PHMB at 20.9 min. 23

24 Cys m/z b Pen m/z NACCys m/z NACPen m/z m/z Figure S-18. Precursor ions scan of a mixture of 1 µm of Cys, Pen, NACCys and NACPen complexes with PHMB. (a) relative abundance of the m/z 355 target ion and retention time of the corresponding precursor compounds (b) combined spectra of parental masses (scanned from 300 to 1500 m/z in negative electrospray ionization) producing the m/z 355 ion over the 30 min chromatogram. 24