Page S-1

Supplementary data

Title of Primary Article:

New finding of nalbuphine metabolites in men: NMR spectroscopy and UPLC–MS/MS

spectrometry assays in a pilot human study

Authors:

Hong-Jaan Wang,1 Cheng-Huei Hsiong,2 Li-Heng Pao,2,3 Wen-Liang Chang,2 Li-Jie Zhang,4 Min-

Jen Lin,2 Shung-Tai Ho,5 Pei-Wei Huang,2 Oliver Yoa-Pu Hu1,2,*

Affiliations:

1 Graduate Institute of Life Sciences, National Defense Medical Center, No.161, Sec. 6, Minquan

E. Rd., Neihu Dist., Taipei City 114, Taiwan

2 School of Pharmacy, National Defense Medical Center, No.161, Sec. 6, Minquan E. Rd., Neihu

Dist., Taipei City 114, Taiwan

3 Research Center for Industry of Human Ecology, Chang Gung University of Science and

Technology, No.261, Wen-hwa 1st Rd., Kwei-shan Township, Taoyuan County, 333, Taiwan

4 National Research Institute of Chinese Medicine, Ministry of Health and Welfare, No. 155-1,

Sec 2, Li Nung St., Beitou Dist., Taipei City 112, Taiwan

5 Department of Anesthesiology, Taipei Veterans General Hospital, No.201, Sec. 2, Shipai Rd.,

Beitou Dist., Taipei City 112, Taiwan

*Corresponding Author:

Hu, O. Y. P.; Phone: +886-2-87923100 ext. 18176; Fax: +886-2-87924859;

E-mail: hyp@ndmctsgh.edu.tw.

Page S-2

The procedures of microsomes incubation and metabolites purification.

NAL (4 mM) was incubated with 24 mg/mL pooled human liver microsomal proteins in 0.1 M

phosphate buffer (pH 7.4) containing 5 mM MgCl2. The reactions was carried out at 37 °C in a

shaking incubator and initiated by adding NADP (20 mM), G6PD (40 IU/mL), G6P (0.2 M) and

UDPGA (75 mM) in a total volume of 25 mL. After a 24 hr incubation, the mixture was alkalized

by the addition of 25 mL 1N Na2CO3 (pH 10) and extracted by a 300 mL addition of extraction

solvent (n-hexane: isopropane = 9: 1, v/v) to remove the unreacted NAL. After discarding the

organic solvent, the liquid layer was then transferred into centrifugal tubes and centrifuged for 10

min at 13,000 g. The supernatants were separately transferred onto preconditioned Oasis HLB 5

mL (200 mg) extraction cartridges (Milford, MA, USA). The cartridges were then washed with 5

mL of water, and the metabolites were eluted with 10 × 5 mL of acetonitrile. The eluates were

separately injected into the HPLC system in a 200 μL injection volume. The collected fractions

containing of each metabolite were then evaporated to dryness under nitrogen gas at 40 ℃, and

the residues were purified by Oasis HLB cartridges following the procedures as described above.

Finally, the crystalline substances were assembled to weigh by microbalance (Mettler Toledo,

Columbus, OH, USA) and subsequently confirmed by qualitative analyses of 1H NMR and mass

spectrometry.

Page S-3

Table S1 1H NMR data of NAL and its metabolites (500 MHz, δ ppm) in D2O.Position NAL 3'-OH NAL 4'-OH NAL N3G N6G

1 6.71 d (8.2) 6.72 d (8.3) 6.72 d (8.3) 6.79 d (8.4) 6.71 d (8.2)2 6.80 d (8.2) 6.82 d (8.2) 6.82 d (8.2) 7.04 d (8.4) 6.82 d (8.2)

5 4.70 ov 4.60 - 4.80 ov 4.60 - 4.80 ov 4.61 d (5.2) 4.89 d (4.5)

6 4.21 m 4.23 m 4.23 m 4.23 m 4.39 m

7α 1.73-1.78 ov 1.78 m 1.77 m 1.64-1.66 ov 1.68-1.77 ov

7β 1.28 m 1.30 m 1.29 m 1.34 m 1.25 m

8α 1.56 m 1.57 m 1.57 m 1.55 m 1.55 m

8β 1.61 m 1.65 m 1.63 m 1.61 m 1.68-1.77 ov

9 3.52 d (6.4) 3.64 d (6.3) 3.53 d (6.4) 3.53-3.57 ov 3.53 d (6.4)

10α 3.08-3.18 ov 3.11 dd (20.1; 6.7) 3.14 d (6.5) 3.08-3.19 ov 3.07-3.13. ov

10β 3.32-3.35 ov 3.30 - 3.37 ov 3.30 - 3.34 ov 3.16- 3.25 ov 3.30-3.35 ov

15α 1.68 dd (14.0; 3.3) 1.71 dd (14.0; 3.6) 1.71 dd (14.1; 3.8) 1.71 dd (13.8; 3.4) 1.68-1.77 ov

15β 2.44 dt (13.4; 5.0) 2.48 dt (13.8; 5.1) 2.48 dt (13.4; 5.2) 2.45 dt (13.7; 5.0) 2.48 dt (13.6; 5.2)

16α 2.85 dt (13.1; 4.1) 2.88 dt (13.1; 4.1) 2.89 dt (13.1; 4.1) 2.85 dt (13.2; 4.1) 2.90 dt (13.2; 4.1)

16β 3.08-3.18ov 3.13 dd (14.1; 4.6) 3.11 m 3.10 dd (13.0; 4.6) 3.07-3.13 ov

1' 3.32-3.35 ov 3.30 - 3.37 ov 3.41 dd (13.6; 7.1) 3.31-3.35 ov 3.29-3.30 ov

3.08-3.18 ov 3.30 - 3.37 ov 3.24 dd (13.4; 9.1) 3.08-3.19 ov 3.18 m

2' 2.67 sep (7.6) 2.45 m 2.69 brs 2.68 sep (7.6) 2.66 sep(7.4)

3' 2.14 brs 3.94 q (8.0) 2.11 m 2.15 brs 2.15 brs

1.78-1.85 ov 2.19 m 1.77-1.86 ov 1.79-1.86 ov

4' 1.93 m 2.23 m 4.39 p (6.7) 1.95 m 1.95 m

1.78-1.85 ov 1.83 m 1.77- 1.86 ov 1.79-1.86 ov

5' 2.07 brs 1.36 m 2.11 m 2.09 brs 2.08 brs

1.78-1.85 ov 1.98 q (9.4) 2.19 m 1.77-1.86 ov 1.79-1.86 ov

Anomeric proton – – – 5.14 d (7.3) 4.61 d (7.9)

Page S-4

Position NAL 3'-OH NAL 4'-OH NAL N3G N6G

2'' – – – 3.53-3.57 ov 3.23 t (8.1)3'' – – – 3.53-3.57 ov 3.42-3.49 ov

4'' – – – 3.53-3.57 ov 3.42-3.49 ov

5'' – – – 3.81 d (6.4) 3.66 d (6.4)

brs = broad singlet; d = doublet; dd = doublet of doublets; dt = doublet of triplets; m = multiplet; ov = overlapped; p = pentet; q = quartet; sep = septet; t = triplet.

The J values, in the brackets, are reported in Hz.

Page S-5

Table S2 Multiple reaction monitoring transitions and fragmentation parameters for the analytes

and internal standard.

AnalytePolarity Precursor

(m/z)Product(m/z)

DP(V)

FP(V)

CEP(V)

CE(eV)

CXP(V)

Retention(min)

NAL ES+ 358.1 340.1 55 269 8 31 6 2.78

3'-OH NAL ES+ 374.1 355.9 59 350 10 40 12 1.39

4'-OH NAL ES+ 374.0 356.2 59 350 10 40 12 1.14

N3G ES+ 534.2 340.3 63 350 10 46 8 1.48

N6G ES+ 534.2 340.3 63 350 10 46 8 2.60

NAX ES+ 328.3 310.3 54 279 7 29 9 1.57

DP: declustering potential; FP: focusing potential; CEP: cell entrance potential; CE: collision energy; CXP: cell exit

potential.

Page S-6

Table S3 Summary of stability tests of NAL and its four metabolites in plasma samples.

Analyte

Stock

solutions

(-20 °C; 30

days)

Working

solution

(25 °C; 4 hr)

Short term

(25 °C; 4 hr)

Autosampler

(4 °C; 24 hr)

Freeze &

Thraw

(3 cycle)

Long term

(-80 °C; 14

days)

NAL 99.2–100.3 94.4–108.9 98.0–107.3 95.6–101.9 103.9–105.8 87.6–104.4

3'-OH NAL 100.9−107.0 104.5–112.1 97.9–109.1 99.7–104.8 95.0–103.8 90.6–99.3

4'-OH NAL 92.3–104.2 100.5–113.6 99.4–105.3 104.5–107.2 102.8–107.7 94.4–106.2

N3G 99.3–101.2 102.6–107.4 90.6–105.3 95.1–99.3 93.9–97.8 86.9–92.6

N6G 96.2–97.3 96.4–110.7 92.4–99.3 97.4–103.7 91.6–106.0 88.5–97.9

NAX 96.7–99.5 98.6–104.5 NA 97.1–99.2 NA NA

Results are expressed as % of control.

Page S-7

Table S4 Pharmacokinetic parameters of NAL and its four metabolites by oral administration of

66 mg NAL in four healthy volunteers.

PK parameter NAL 3'-OH NAL 4'-OH NAL N3G N6G

Cmax (ng/mL) 21.5 ± 4.2 38.9 ± 14.4 58.6 ± 14.9 1147.0 ± 368.8 89.5 ± 24.5

Tmax (hr) 0.6 ± 0.3 0.5 ± 0.2 1.1 ± 0.5 0.9 ± 0.4 1.2 ± 0.4

AUCt

(hr*ng/mL) 73.4 ± 15.5 196.5 ± 37.5 449.1 ± 113.6 3547.7 ± 279.0 326.9 ± 30.6

AUC∞

(hr*ng/mL) 89.5 ± 29.3 263.8 ± 65.4 593.3 ± 194.5 3828.8 ± 327.8 347.7 ± 43.5

T1/2 (hr) 8.9 ± 1.8 13.2 ± 2.9 11.9 ± 1.9 8.5 ± 3.5 7.6 ± 2.9

k (1/hr) 0.1 ± 0.0 0.1 ± 0.0 0.1 ± 0.0 0.1 ± 0.1 0.1 ± 0.0

CL/F (L/hr) 807.9 ± 298.6 262.1 ± 62.9 119.2 ± 32.9 17.3 ± 1.5 192.0 ± 23.2

Vd/F (L) 10075.9 ± 2742.4 4908.5 ± 1276.3 1999.7 ± 471.8 211.2 ± 82.1 2037.8 ± 571.8

Data are shown as mean ± SD

Page S-8

Fig. S1-1 1H NMR spectrum of NAL (500 MHz, D2O, 25 °C).

Page S-9

ppm

8 7 6 5 4 3 2 1 ppm8

7

6

5

4

3

2

1NAME huyp-wanghj-13Oct10-nalEXPNO 74PROCNO 1Date_ 20131011Time 7.30INSTRUM spectPROBHD 5 mm CPPBBO BBPULPROG cosygpppqf45TD 2048SOLVENT D2ONS 4DS 16SWH 6510.417 HzFIDRES 3.178914 HzAQ 0.1573364 secRG 1030DW 76.800 usecDE 22.00 usecTE 298.0 KD0 0.00000300 secD1 1.00000000 secD11 0.03000000 secD12 0.00002000 secD13 0.00000400 secD16 0.00020000 secIN0 0.00015360 sec

======== CHANNEL f1 ========SFO1 500.1730010 MHzNUC1 1HP0 5.85 usecP1 11.70 usecP17 2500.00 usecND0 1TD 512SFO1 500.173 MHzFIDRES 12.715657 HzSW 13.016 ppmFnMODE QFSI 1024SF 500.1699974 MHzWDW QSINESSB 0LB 0.00 HzGB 0PC 1.00SI 1024MC2 QFSF 500.1699972 MHzWDW QSINESSB 0LB 0.00 HzGB 0

500MHz COSY D2O NAL

Fig. S1-2 1H-1H COSY spectrum of NAL (500 MHz, D2O, 25 °C).

Page S-10

Page S-11

ppm

8 7 6 5 4 3 2 1 ppm8

7

6

5

4

3

2

1NAME huyp-wanghj-13Oct10-nalEXPNO 73PROCNO 1Date_ 20131011Time 4.33INSTRUM spectPROBHD 5 mm CPPBBO BBPULPROG noesyphps.casTD 2048SOLVENT D2ONS 8DS 16SWH 7002.801 HzFIDRES 3.419337 HzAQ 0.1462772 secRG 22.6DW 71.400 usecDE 10.00 usecTE 298.0 KD0 0.00005650 secD1 2.00000000 secD8 0.40000001 secD11 0.03000000 secD12 0.00002000 secD13 0.00000400 secIN0 0.00014280 secL6 20L7 4ZGOPTNS

======== CHANNEL f1 ========SFO1 500.1730010 MHzNUC1 1HP1 11.70 usecP18 100000.00 usecND0 1TD 512SFO1 500.173 MHzFIDRES 13.677346 HzSW 14.001 ppmFnMODE States-TPPISI 1024SF 500.1699946 MHzWDW QSINESSB 2LB 0.00 HzGB 0PC 1.00SI 1024MC2 States-TPPISF 500.1699966 MHzWDW QSINESSB 2LB 0.00 HzGB 0

500MHz NOESY D2O NAL

Fig. S1-3 NOESY spectrum of NAL (500 MHz, D2O, 25 °C).

Page S-12

Fig. S1-4 13C-NMR spectrum of NAL obtained with sample spinning and proton decoupling (500 MHz, 25 °C) in D2O.

Page S-13

ppm

8.0 7.5 7.0 6.5 6.0 5.5 5.0 4.5 4.0 3.5 3.0 2.5 2.0 1.5 1.0 0.5 ppm

140

120

100

80

60

40

20

NAME huyp-wanghj-13Oct10-nalEXPNO 72PROCNO 1Date_ 20131011Time 3.15INSTRUM spectPROBHD 5 mm CPPBBO BBPULPROG hsqcedetgpsisp2.3TD 1024SOLVENT D2ONS 8DS 16SWH 6009.615 HzFIDRES 5.868765 HzAQ 0.0852468 secRG 32DW 83.200 usecDE 35.00 usecTE 298.0 KCNST2 155.0000000CNST17 -0.5000000D0 0.00000300 secD1 1.00000000 secD4 0.00161290 secD11 0.03000000 secD16 0.00020000 secD21 0.00322581 secD24 0.00080645 secIN0 0.00001660 sec

======== CHANNEL f1 ========SFO1 500.1725009 MHzNUC1 1HP1 11.70 usecP2 23.40 usecP28 0.00 usecND0 2TD 512SFO1 125.7779 MHzFIDRES 58.829067 HzSW 239.474 ppmFnMODE Echo-AntiechoSI 2048SF 500.1699991 MHzWDW QSINESSB 2LB 0.00 HzGB 0PC 1.00SI 2048MC2 echo-antiechoSF 125.7676985 MHzWDW QSINESSB 2LB 0.00 HzGB 0

500MHz HSQC D2O NAL

Fig. S1-5 HSQC spectrum of NAL (500 MHz, D2O, 25 °C)

Page S-14

.

ppm

8.0 7.5 7.0 6.5 6.0 5.5 5.0 4.5 4.0 3.5 3.0 2.5 2.0 1.5 1.0 0.5 ppm

140

120

100

80

60

40

20

NAME huyp-wanghj-13Oct10-nalEXPNO 71PROCNO 1Date_ 20131010Time 21.34INSTRUM spectPROBHD 5 mm CPPBBO BBPULPROG hmbcetgpl3ndTD 2048SOLVENT D2ONS 32DS 16SWH 6250.000 HzFIDRES 3.051758 HzAQ 0.1638900 secRG 2050DW 80.000 usecDE 10.00 usecTE 298.0 KCNST6 120.0000000CNST7 170.0000000CNST13 8.0000000CNST30 0.5981125D0 0.00000300 secD1 1.00000000 secD6 0.06250000 secD16 0.00020000 secIN0 0.00001660 sec

======== CHANNEL f1 ========SFO1 500.1730010 MHzNUC1 1HP1 11.70 usecP2 23.40 usecND0 2TD 512SFO1 125.7817 MHzFIDRES 58.829067 HzSW 239.466 ppmFnMODE Echo-AntiechoSI 4096SF 500.1700009 MHzWDW QSINESSB 2LB 0.00 HzGB 0PC 1.00SI 2048MC2 echo-antiechoSF 125.7677144 MHzWDW QSINESSB 2LB 0.00 HzGB 0

500MHz HMBC D2O NAL

Fig. S1-6 HMBC spectrum of NAL (500 MHz, D2O, 25 °C).

Page S-15

Fig. S2-1 1H NMR spectrum of 3'-OH NAL (500 MHz, D2O, 25 °C).

Page S-16

ppm

9 8 7 6 5 4 3 2 1 ppm9

8

7

6

5

4

3

2

1 NAME changwl-wanghc-10Apr08-2EXPNO 82PROCNO 1Date_ 20100408Time 14.52INSTRUM spectPROBHD 5 mm CPTCI 1H-PULPROG cosygpqfTD 2048SOLVENT D2ONS 4DS 4SWH 9615.385 HzFIDRES 4.695012 HzAQ 0.1065460 secRG 645DW 52.000 usecDE 25.00 usecTE 298.0 KD0 0.00000300 secD1 1.00000000 secD13 0.00000400 secD16 0.00010000 secIN0 0.00010400 sec

======== CHANNEL f1 ========NUC1 1HP0 4.50 usecP1 9.00 usecPL1 3.50 dBPL1W 10.51331329 WSFO1 800.2040010 MHz

====== GRADIENT CHANNEL =====GPNAM1 SINE.100GPZ1 10.00 %P16 1000.00 usecND0 1TD 512SFO1 800.204 MHzFIDRES 18.780048 HzSW 12.016 ppmFnMODE QFSI 1024SF 800.2000004 MHzWDW SINESSB 0LB 0.00 HzGB 0PC 1.00SI 1024MC2 QFSF 800.2000004 MHzWDW SINESSB 0LB 0.00 HzGB 0

800MHz COSY D2O 2

NTU

Fig. S2-2 1H-1H COSY spectrum of 3′-OH NAL (500 MHz, D2O, 25 °C).

Page S-17

ppm

9 8 7 6 5 4 3 2 1 ppm9

8

7

6

5

4

3

2

1 NAME changwl-wanghc-10Apr08-2EXPNO 81PROCNO 1Date_ 20100408Time 11.46INSTRUM spectPROBHD 5 mm CPTCI 1H-PULPROG noesygpph19TD 2048SOLVENT D2ONS 8DS 4SWH 9615.385 HzFIDRES 4.695012 HzAQ 0.1065460 secRG 645DW 52.000 usecDE 25.00 usecTE 298.0 KD0 0.00004054 secD1 2.00000000 secD8 0.60000002 secD12 0.00002000 secD16 0.00010000 secD19 0.00010400 secIN0 0.00010400 sec

======== CHANNEL f1 ========NUC1 1HP0 9.00 usecP1 9.00 usecP27 9.00 usecPL1 3.50 dBPL18 3.50 dBPL1W 10.51331329 WPL18W 10.51331329 WSFO1 800.2037640 MHz

====== GRADIENT CHANNEL =====GPNAM1 SINE.100GPZ1 20.00 %P16 1000.00 usecND0 1TD 512SFO1 800.2038 MHzFIDRES 18.780043 HzSW 12.016 ppmFnMODE TPPISI 1024SF 800.1999971 MHzWDW QSINESSB 2LB 0.00 HzGB 0PC 1.00SI 1024MC2 TPPISF 800.1999971 MHzWDW QSINESSB 2LB 0.00 HzGB 0

800MHz NOESY D2O 2

NTU

Fig. S2-3 NOESY spectrum of 3′-OH NAL (500 MHz, D2O, 25 °C).

Page S-18

Fig. S3-1 1H NMR spectrum of 4'-OH NAL (500 MHz, D2O, 25 °C).

Page S-19

ppm

9 8 7 6 5 4 3 2 1 ppm9

8

7

6

5

4

3

2

1 NAME changwl-wanghc-10Apr08-3EXPNO 82PROCNO 1Date_ 20100408Time 19.44INSTRUM spectPROBHD 5 mm CPTCI 1H-PULPROG cosygpqfTD 2048SOLVENT D2ONS 4DS 4SWH 9615.385 HzFIDRES 4.695012 HzAQ 0.1065460 secRG 101DW 52.000 usecDE 25.00 usecTE 298.0 KD0 0.00000300 secD1 1.00000000 secD13 0.00000400 secD16 0.00010000 secIN0 0.00010400 sec

======== CHANNEL f1 ========NUC1 1HP0 4.50 usecP1 9.00 usecPL1 3.50 dBPL1W 10.51331329 WSFO1 800.2040010 MHz

====== GRADIENT CHANNEL =====GPNAM1 SINE.100GPZ1 10.00 %P16 1000.00 usecND0 1TD 512SFO1 800.204 MHzFIDRES 18.780048 HzSW 12.016 ppmFnMODE QFSI 1024SF 800.1999996 MHzWDW SINESSB 0LB 0.00 HzGB 0PC 1.00SI 1024MC2 QFSF 800.2000007 MHzWDW SINESSB 0LB 0.00 HzGB 0

800MHz COSY D2O 3

NTU

Fig. S3-2 1H-1H COSY spectrum of 4′-OH NAL (500 MHz, D2O, 25 °C).

Page S-20

ppm

9 8 7 6 5 4 3 2 1 ppm9

8

7

6

5

4

3

2

1 NAME changwl-wanghc-10Apr08-3EXPNO 81PROCNO 1Date_ 20100408Time 16.38INSTRUM spectPROBHD 5 mm CPTCI 1H-PULPROG noesygpph19TD 2048SOLVENT D2ONS 8DS 4SWH 9615.385 HzFIDRES 4.695012 HzAQ 0.1065460 secRG 287DW 52.000 usecDE 25.00 usecTE 298.0 KD0 0.00004054 secD1 2.00000000 secD8 0.60000002 secD12 0.00002000 secD16 0.00010000 secD19 0.00010400 secIN0 0.00010400 sec

======== CHANNEL f1 ========NUC1 1HP0 9.00 usecP1 9.00 usecP27 9.00 usecPL1 3.50 dBPL18 3.50 dBPL1W 10.51331329 WPL18W 10.51331329 WSFO1 800.2037640 MHz

====== GRADIENT CHANNEL =====GPNAM1 SINE.100GPZ1 20.00 %P16 1000.00 usecND0 1TD 512SFO1 800.2038 MHzFIDRES 18.780043 HzSW 12.016 ppmFnMODE TPPISI 1024SF 800.1999995 MHzWDW QSINESSB 2LB 0.00 HzGB 0PC 1.00SI 1024MC2 TPPISF 800.2000002 MHzWDW QSINESSB 2LB 0.00 HzGB 0

800MHz NOESY D2O 3

NTU

Fig. S3-3 NOESY spectrum of 4′-OH NAL (500 MHz, D2O, 25 °C).

Page S-21

Fig. S4-1 1H NMR spectrum of N3G (500 MHz, D2O, 25 °C).

Page S-22

ppm

8 7 6 5 4 3 2 1 ppm8

7

6

5

4

3

2

1NAME huyp-wanghj-13Oct09-n3gEXPNO 74PROCNO 1Date_ 20131010Time 12.21INSTRUM spectPROBHD 5 mm CPPBBO BBPULPROG cosygpppqf45TD 2048SOLVENT D2ONS 4DS 16SWH 6510.417 HzFIDRES 3.178914 HzAQ 0.1573364 secRG 912DW 76.800 usecDE 22.00 usecTE 288.0 KD0 0.00000300 secD1 1.00000000 secD11 0.03000000 secD12 0.00002000 secD13 0.00000400 secD16 0.00020000 secIN0 0.00015360 sec

======== CHANNEL f1 ========SFO1 500.1730010 MHzNUC1 1HP0 5.80 usecP1 11.60 usecP17 2500.00 usecND0 1TD 512SFO1 500.173 MHzFIDRES 12.715657 HzSW 13.016 ppmFnMODE QFSI 1024SF 500.1699951 MHzWDW QSINESSB 0LB 0.00 HzGB 0PC 1.00SI 1024MC2 QFSF 500.1699950 MHzWDW QSINESSB 0LB 0.00 HzGB 0

500MHz COSY D2O N3G

Fig. S4-2 1H-1H COSY of N3G (500 MHz, D2O, 25 °C).

Page S-23

ppm

8 7 6 5 4 3 2 1 ppm8

7

6

5

4

3

2

1NAME huyp-wanghj-13Oct09-n3gEXPNO 63PROCNO 1Date_ 20131013Time 14.18INSTRUM spectPROBHD 5 mm CPPBBO BBPULPROG noesyphps.casTD 2048SOLVENT D2ONS 16DS 32SWH 7002.801 HzFIDRES 3.419337 HzAQ 0.1462772 secRG 32DW 71.400 usecDE 10.00 usecTE 298.0 KD0 0.00005778 secD1 2.00000000 secD8 0.40000001 secD11 0.03000000 secD12 0.00002000 secD13 0.00000400 secIN0 0.00014280 secL6 20L7 4ZGOPTNS

======== CHANNEL f1 ========SFO1 500.1730010 MHzNUC1 1HP1 10.70 usecP18 100000.00 usecND0 1TD 512SFO1 500.173 MHzFIDRES 13.677346 HzSW 14.001 ppmFnMODE States-TPPISI 1024SF 500.1699978 MHzWDW QSINESSB 2LB 0.00 HzGB 0PC 1.00SI 1024MC2 States-TPPISF 500.1700001 MHzWDW QSINESSB 2LB 0.00 HzGB 0

500MHz NOESY D2O N3G

Fig. S4-3 NOESY spectrum of N3G (500 MHz, D2O, 25 °C).

Page S-24

Fig. S4-4 13C-NMR spectrum of N3G obtained with sample spinning and proton decoupling (500 MHz, 25 °C) in D2O.

H1′′

C1′′

Page S-25

ppm

8.0 7.5 7.0 6.5 6.0 5.5 5.0 4.5 4.0 3.5 3.0 2.5 2.0 1.5 1.0 0.5 ppm

140

120

100

80

60

40

20

NAME huyp-wanghj-13Oct09-n3gEXPNO 72PROCNO 1Date_ 20131010Time 5.05INSTRUM spectPROBHD 5 mm CPPBBO BBPULPROG hsqcedetgpsisp2.3TD 1024SOLVENT D2ONS 8DS 16SWH 6009.615 HzFIDRES 5.868765 HzAQ 0.0852468 secRG 64DW 83.200 usecDE 35.00 usecTE 288.0 KCNST2 155.0000000CNST17 -0.5000000D0 0.00000300 secD1 1.00000000 secD4 0.00161290 secD11 0.03000000 secD16 0.00020000 secD21 0.00322581 secD24 0.00080645 secIN0 0.00001660 sec

======== CHANNEL f1 ========SFO1 500.1725009 MHzNUC1 1HP1 11.60 usecP2 23.20 usecP28 0.00 usecND0 2TD 512SFO1 125.7779 MHzFIDRES 58.829067 HzSW 239.474 ppmFnMODE Echo-AntiechoSI 2048SF 500.1699977 MHzWDW QSINESSB 2LB 0.00 HzGB 0PC 1.00SI 2048MC2 echo-antiechoSF 125.7677067 MHzWDW QSINESSB 2LB 0.00 HzGB 0

500MHz HSQC D2O N3G

Fig. S4-5 HSQC spectrum of N3G (500 MHz, D2O, 25 °C).

H1′′

C3

Page S-26

ppm

8.0 7.5 7.0 6.5 6.0 5.5 5.0 4.5 4.0 3.5 3.0 2.5 2.0 1.5 1.0 0.5 ppm

180

160

140

120

100

80

60

40

20

NAME huyp-wanghj-13Oct09-n3gEXPNO 71PROCNO 1Date_ 20131009Time 23.25INSTRUM spectPROBHD 5 mm CPPBBO BBPULPROG hmbcetgpl3ndTD 2048SOLVENT D2ONS 32DS 16SWH 6250.000 HzFIDRES 3.051758 HzAQ 0.1638900 secRG 2050DW 80.000 usecDE 10.00 usecTE 288.0 KCNST6 120.0000000CNST7 170.0000000CNST13 8.0000000CNST30 0.5981125D0 0.00000300 secD1 1.00000000 secD6 0.06250000 secD16 0.00020000 secIN0 0.00001660 sec

======== CHANNEL f1 ========SFO1 500.1730010 MHzNUC1 1HP1 11.60 usecP2 23.20 usecND0 2TD 512SFO1 125.7817 MHzFIDRES 58.829067 HzSW 239.466 ppmFnMODE Echo-AntiechoSI 2048SF 500.1699980 MHzWDW QSINESSB 2LB 0.00 HzGB 0PC 1.00SI 2048MC2 echo-antiechoSF 125.7677133 MHzWDW QSINESSB 2LB 0.00 HzGB 0

500MHz HMBC D2O N3G

Fig. S4-6 HMBC spectrum of N3G (500 MHz, D2O, 25 °C).

Page S-27

Fig. S5-1 1H NMR spectrum of N6G (500 MHz, D2O, 25 °C).

Page S-28

ppm

8 7 6 5 4 3 2 1 ppm8

7

6

5

4

3

2

1NAME huyp-wanghj-13Oct08-n6gEXPNO 74PROCNO 1Date_ 20131009Time 8.16INSTRUM spectPROBHD 5 mm CPPBBO BBPULPROG cosygpppqf45TD 2048SOLVENT D2ONS 4DS 16SWH 6510.417 HzFIDRES 3.178914 HzAQ 0.1573364 secRG 1440DW 76.800 usecDE 22.00 usecTE 298.0 KD0 0.00000300 secD1 1.00000000 secD11 0.03000000 secD12 0.00002000 secD13 0.00000400 secD16 0.00020000 secIN0 0.00015360 sec

======== CHANNEL f1 ========SFO1 500.1730010 MHzNUC1 1HP0 5.68 usecP1 11.35 usecP17 2500.00 usecND0 1TD 512SFO1 500.173 MHzFIDRES 12.715657 HzSW 13.016 ppmFnMODE QFSI 2048SF 500.1699989 MHzWDW QSINESSB 0LB 0.00 HzGB 0PC 1.00SI 2048MC2 QFSF 500.1699992 MHzWDW QSINESSB 0LB 0.00 HzGB 0

500MHz COSY D2O N6G

Fig. S5-2 1H-1H COSY spectrum of N6G (500 MHz, D2O, 25 °C).

Page S-29

ppm

8 7 6 5 4 3 2 1 ppm8

7

6

5

4

3

2

1NAME huyp-wanghj-13Oct08-n6gEXPNO 73PROCNO 1Date_ 20131009Time 5.17INSTRUM spectPROBHD 5 mm CPPBBO BBPULPROG noesyphprTD 2048SOLVENT D2ONS 8DS 16SWH 6009.615 HzFIDRES 2.934382 HzAQ 0.1704436 secRG 25.4DW 83.200 usecDE 10.00 usecTE 298.0 KD0 0.00006875 secD1 2.00000000 secD8 0.40000001 secD11 0.03000000 secD12 0.00002000 secD13 0.00000400 secIN0 0.00016640 sec

======== CHANNEL f1 ========SFO1 500.1723514 MHzNUC1 1HP1 11.35 usecND0 1TD 512SFO1 500.1724 MHzFIDRES 11.737530 HzSW 12.015 ppmFnMODE States-TPPISI 2048SF 500.1699997 MHzWDW QSINESSB 2LB 0.00 HzGB 0PC 1.00SI 2048MC2 States-TPPISF 500.1700006 MHzWDW QSINESSB 2LB 0.00 HzGB 0

500MHz NOESY D2O N6G

Fig. S5-3 NOESY spectrum of N6G (500 MHz, D2O, 25 °C).

Page S-30

Fig. S5-4 13C-NMR spectrum of N6G obtained with sample spinning and proton decoupling (500 MHz, 25 °C) in D2O.

H1′′

C1′′

Page S-31

ppm

8.0 7.5 7.0 6.5 6.0 5.5 5.0 4.5 4.0 3.5 3.0 2.5 2.0 1.5 1.0 0.5 ppm

180

160

140

120

100

80

60

40

20

NAME huyp-wanghj-13Oct08-n6gEXPNO 72PROCNO 1Date_ 20131009Time 3.22INSTRUM spectPROBHD 5 mm CPPBBO BBPULPROG hsqcedetgpsisp2.3TD 1024SOLVENT D2ONS 12DS 16SWH 6009.615 HzFIDRES 5.868765 HzAQ 0.0852468 secRG 64DW 83.200 usecDE 35.00 usecTE 298.0 KCNST2 155.0000000CNST17 -0.5000000D0 0.00000300 secD1 1.00000000 secD4 0.00161290 secD11 0.03000000 secD16 0.00020000 secD21 0.00322581 secD24 0.00080645 secIN0 0.00001660 sec

======== CHANNEL f1 ========SFO1 500.1725009 MHzNUC1 1HP1 11.35 usecP2 22.70 usecP28 0.00 usecND0 2TD 512SFO1 125.7779 MHzFIDRES 58.829067 HzSW 239.474 ppmFnMODE Echo-AntiechoSI 2048SF 500.1699983 MHzWDW QSINESSB 2LB 0.00 HzGB 0PC 1.00SI 2048MC2 echo-antiechoSF 125.7677099 MHzWDW QSINESSB 2LB 0.00 HzGB 0

500MHz HSQC D2O N6G

Fig. S5-5 HSQC spectrum of N6G (500 MHz, D2O, 25 °C).

H1′′

C6

Page S-32

ppm

8.0 7.5 7.0 6.5 6.0 5.5 5.0 4.5 4.0 3.5 3.0 2.5 2.0 1.5 1.0 0.5 ppm

180

160

140

120

100

80

60

40

20

NAME huyp-wanghj-13Oct08-n6gEXPNO 71PROCNO 1Date_ 20131008Time 21.41INSTRUM spectPROBHD 5 mm CPPBBO BBPULPROG hmbcetgpl3ndTD 2048SOLVENT D2ONS 32DS 16SWH 6250.000 HzFIDRES 3.051758 HzAQ 0.1638900 secRG 2050DW 80.000 usecDE 10.00 usecTE 298.0 KCNST6 120.0000000CNST7 170.0000000CNST13 8.0000000CNST30 0.5981125D0 0.00000300 secD1 1.00000000 secD6 0.06250000 secD16 0.00020000 secIN0 0.00001660 sec

======== CHANNEL f1 ========SFO1 500.1730010 MHzNUC1 1HP1 11.35 usecP2 22.70 usecND0 2TD 512SFO1 125.7817 MHzFIDRES 58.829067 HzSW 239.466 ppmFnMODE Echo-AntiechoSI 2048SF 500.1699992 MHzWDW QSINESSB 2LB 0.00 HzGB 0PC 1.00SI 2048MC2 echo-antiechoSF 125.7677110 MHzWDW QSINESSB 2LB 0.00 HzGB 0

500MHz HMBC D2O N6G

Fig. S5-6 HMBC spectrum N6G (500 MHz, D2O, 25 °C).

Page S-33

Fig. S6 Q3 scan spectra showing product ion [M+H]+ of (a) NAL, (b) naloxone, (c) 3'-OH NAL,

(d) 4'-OH NAL, (e) N3G and (f) N6G.

Page S-34

Fig. S7 MRM chromatograms of (a) blank human plasma sample; (b) blank human plasma

sample spiked with NAL and its metabolites at LLOQ; (c) plasma sample from one subject at 2 h

after 66 mg NAL oral administered.