Table S1. Optimized MRM transitions and compound parameters for the analysis of DRV, RTV,
TNF and alprazolam (IS).
Compound
Transition (m/z)
mode
Cone voltage (V)
Collision Energy (eV)
Ionization
DRV
547.9/391.8
20
13
ES+
RTV
721.5/295.9
30
18
ES+
TNF
288.3/175.9
30
25
ES+
Alprazolam (IS)
308.4/273.6
45
20
ES+
Table S2. Calibration standards and quality control sample concentrations of DRV, RTV and
TNF.
Standards and QCs
DRV (ng/mL)
RTV (ng/mL)
TNF (ng/mL)
STD 1
20.0
2.0
14.0
STD 2
40.0
4.0
28.0
STD 3
100
20
50
STD 4
400
60
100
STD 5
1000
120
200
STD 6
2500
480
400
STD 7
5000
960
650
STD 8
8000
1920
1200
STD 9
12000
2280
1600
LQC
60.0
6.0
40
MQC
4500
1000
650
HQC
9000
2000
1300
Table S3. Extraction recoveries of DRV, RTV, TNF and IS with different organic solvents at
LOQ and HQC concentrationsa.
Solvent
a
DRV
RTV
TNF
Alprazolam (IS)
Ethyl acetate
64.37±7.3
78.41±5.2
60.12±5.8
59.42±7.4
Methyl tert-butyl ether
83.42±2.9
82.92±4.2
86.44±2.4
80.71±4.2
n-hexane
87.51±3.70
59.53±6.2
54.31±6.2
74.83±3.9
Diethyl ether
64.88±5.3
80.44±5.3
58.75±5.8
60.42±4.3
n-hexane–isopropanol
72.52±3.8
61.93± 3.2
57.32±5.5
75.85±3.4
n-hexane–ethyl acetate
73.91±4.6
50.00±5.7
69.70±4.4
70.34±4.6
n= three determinations
Table S4. Summary of stability data of DRV, RTV and TNF in human plasma.
LQC (ng/mL)
Stability
MQC (ng/mL)
HQC (ng/mL)
DRV
RTV
TNF
DRV
RTV
TNF
DRV
RTV
TNF
60.0
6.0
40.0
4500
1000
650
9000
2000
1300
2092.41
1348.82
Room temp(12h)
Mean conc (n=3)
50.89
5.58
41.29
4538.25 1040.35
659.79
8667.00
CV (%)
2.71
3.41
3.14
3.11
5.22
2.81
5.23
3.21
3.94
Bias (%)
-1.84
-7.00
3.22
0.85
-4.01
-1.50
-3.33
-4.62
-3.75
60.13
5.71
38.49
4436.10
973.26
662.72
CV (%)
3.22
1.49
4.21
4.11
2.41
3.17
2.99
4.92
2.98
Bias (%)
2.16
-4.83
-3.77
-1.42
-2.67
1.95
3.43
-1.08
-2.30
60.09
5.86
40.88
4474.35
1031.77
CV (%)
1.33
2.61
2.78
3.23
3.14
2.19
3.81
4.11
2.83
Bias (%)
1.50
-2.32
2.20
-0.57
-5.19
2.90
-1.94
2.54
4.30
60.21
5.63
40.61
4613.36
967.29
693.28
9117.00
2013.13 1332.40
CV (%)
2.66
4.96
2.69
3.21
4.54
2.77
4.61
Bias (%)
3.50
-6.15
1.52
2.52
4.57
6.66
1.13
50.82
6.15
38.69
4437.90
1022.80
671.42
8695.80
CV (%)
2.73
3.81
1.88
4.11
3.21
4.00
3.44
4.62
4.63
Bias (%)
-3.00
2.50
-3.27
-1.28
1.02
3.29
-3.28
1.065
-2.99
Auto sampler(48h)
Mean conc (n=3)
9308.70 1978.37 1270.0 0
Short-term (12h)
Mean conc (n=3)
668.88
8825.40 2050.88 1356. 31
Freeze-thaw
Mean conc (n=3)
3.81
0.65
2.38
-1.30
Long-term
Mean conc (n=3)
2021.30 1309.54
Table S5. System suitability and dilution integrity results of DRV, RTV and TNF drugs.
DRV
RTV
TNF
4500
1000
650
4351.23
1014.70
618.42
96.70±2.61
101.47±0.91
95.14±1.38
18000
4000
2600
4500
1000
650
4521.33
1020.70
658.16
100.47±0.88
102.07±2.11
101.25±1.44
System suitability
Theoretical conc. (ng/mL)
a
Experimental conc. (ng/mL)
%Recovery±RSD
Dilution Integrity
Theoretical conc. (ng/mL)
¼ Dilution (ng/mL)
b
Experimental conc. (ng/mL)
%Recovery±RSD
a
b
average concentration of six determinations
average concentration of three determinations
Table S6. Pharmacokinetic data of DRV, RTV and TNF after their oral administration in 8
healthy male volunteers.
Parameter
DRV
RTV
TNF
Cmax (ng/mL)
5870 ± 1618
820 ± 345
346 ± 133
Tmax (hr)
3.87 ± 1.86
3.07 ± 1.48
1.50 ± 0.40
t1/2
15.13 ± 7.98
5.26 ±2.48
14.2±8.41
82550 ± 33491
5275 ± 2640
2569 ± 690
87330 ± 37143
5620± 2140
3624±1630
(hr)
AUC0-24
(ng.hr/mL)
AUC0-∞ (ng.hr/mL)
Cmax: maximum plasma concentration.
Tmax: time point of maximum plasma concentration.
t1/2: half life of drug elimination during the terminal phase.
AUC0–24: area under the plasma concentration-time curve from zero hour to 24 hr.
AUC0–∞: area under the plasma concentration-time curve from zero hour to infinity.
Figure S1. Chemical structures of a) DRV b) RTV and c) TNF and d) Alprazolam.
Figure S2. Representative mass spectra of DRV, RTV, TNF and Alprazolam drugs in positive
ESI mode.
Figure S3. Representative chromatogram for subject sample I) DRV II) RTV III) TNF and IV)
Alprazolam after 1.0 hr of oral dosage.