Upload
others
View
9
Download
0
Embed Size (px)
Citation preview
Test report Number 5144 G
Object of analysis: 5144 G
Customer: Samples: 1
Batch: Arrived on: 2020/01/02
Subject: Identification, other ingredients, quantification
Method(s): NMR (nuclear magnetic resonance spectroscopy) qNMR (quantitative NMR)
Instrumentation: Bruker Avance III 700 MHz NMR spectrometer Mettler Toledo ME204 analytical balance
Sample / Analysis Method Status Result Value
5144 G:
Identification NMR N Oxandrolone [53-39-4] Identified
Other ingredients NMR N Not identified
Quantification qNMR N Component
Oxandrolone
Method status: G = GMP A = accreditated V = generally validated P = validated on product N = not validated E = external lab
Remarks: In this sample the steroid component was determined to be Oxandrolone. The other components in the tablets were not soluble in chloroform.
Signatures: Date: 2020/03/26
7.26%
72.6 mg/g 25.4 mg/tablet
For the qNMR experiment, 128 scans were acquired, recycling delay was set to 20 seconds, 90° pulse was used.
Compound [CAS number] Molar mass Weight taken
Chemical
shift of
chosen ¹H
NMR signal
Integral of that
¹H NMR signal
Number of H's
under that
signal
Content in
the powder
(mass%)
Content in
the
powder
(mg/g)
Content in the
tablet
(mg/tablet)
Reference for the calculation method:
https://doi.org/10.1016/j.jpba.2010.02.007
A tablet (0.3495 g) of the "5144 G" sample was crushed with a mortar and pestle, and a portion (0.1376 g) of the obtained white powder was washed with
CDCl3 (3×0.7 mL + 0.3 mL), the liquid was each time centrifuged and filtered into an NMR tube and 0.0425 g of 1,4-dimethoxybenzene (99+% purity) was
weighed into the same NMR tube. Shaked, giving a clear homogeneous solution. In order to verify that all soluble (soluble in CDCl3) components have been
washed into the NMR tube, the filter was washed once more with 0.7 mL of CDCl3 and the liquid was collected into a separate NMR tube. ¹H NMR spectrum
measurement of the 2nd
tube confirmed that all of the soluble components had been dissolved into the 1st
NMR tube as there were no signals for the steroid
component visible.
In this sample the steroid component was determined to be Oxandrolone. The other components in the tablets were not soluble in chloroform.
https://en.wikipedia.org/wiki/Oxandrolone
Rundlöf, T.; Mathiasson, M.; Bekiroglu, S.; Hakkarainen, B.; Bowden, T.; Arvidsson, T. Survey and qualification of internal standards for quantification by ¹H
NMR spectroscopy, J. Pharm. Biomed. Anal. 2010, 52, 5, 645-651.
Quantitative analysis of the "5144 G" sample
Oxandrolone [53-39-4] 306.45 g/mol 0.1376 g 4.23 ppm 1.0000 1 7.26% 72.6 mg/g 25.4 mg/tablet
1,4-Dimethoxybenzene [150-78-7] 138.17 g/mol 0.0425 g 6.84 ppm 12.5684 4 99.0%
Photo of the sample
5144 G (white tablets)
10.0 9.5 9.0 8.5 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 0.0 ppm
1.300
1.307
1.344
1.348
1.357
1.363
1.368
1.441
1.446
1.452
1.458
1.467
1.473
1.477
1.483
1.492
1.497
1.502
1.505
1.511
1.519
1.524
1.528
1.574
1.580
1.582
1.593
1.598
1.611
1.616
1.685
1.693
1.699
1.713
1.722
1.727
1.733
1.742
1.746
1.756
1.760
1.764
1.795
1.800
1.816
2.207
2.226
2.234
2.253
2.501
2.509
2.528
2.536
3.771
3.914
3.929
4.227
4.242
6.841
7.260
1.1
524
3.0
425
1.1
382
2.9
977
5.3
300
3.4
470
1.2
407
4.5
688
5.1
689
3.1
971
1.0
355
1.0
216
1.0
006
18.9
123
0.9
948
1.0
000
12.5
684
Current Data ParametersNAME Sample-5144-G-DiMeOBn_2020-03-16_CDCl3EXPNO 12
PROCNO 1
F2 - Acquisition Parameters
Date_ 20200316Time 16.02INSTRUM spect
PROBHD 5 mm PABBO BB-PULPROG zgTD 131072
SOLVENT CDCl3NS 128DS 4
SWH 12626.263 HzFIDRES 0.096331 HzAQ 5.1904511 sec
RG 144DW 39.600 usecDE 6.50 usec
TE 298.2 KD1 20.00000000 secTD0 1
======== CHANNEL f1 ========SFO1 700.0833254 MHz
NUC1 1HP1 15.74 usecPLW1 26.00000000 W
F2 - Processing parametersSI 262144
SF 700.0800301 MHzWDW EMSSB 0
LB 0.10 HzGB 0PC 1.00
TMS
CHCl3
1,4
-dim
eth
oxybenzene
1,4
-dim
eth
oxybenzene
10
5
1
4
O
3
9
8
6
7
11
12
14
13
15
16
17
O
19
1820
OH
H H
H
H
Oxandrolone
qNMRSample:in CDCl3
qNMR of tablet (0.3495 g tablet, 0.1376 g sample) of "5144 G" extracted with CDCl3 (3×0.7 mL+0.3 mL) + 0.0425 g of 1,4-dimethoxybenzene
11H
5.65.75.85.96.06.16.26.36.46.56.66.76.86.97.07.17.27.37.47.57.67.77.87.9 ppm
6.841
7.260
12.5
684
CHCl3
1,4
-dim
eth
oxybenzene
Expansion
qNMRSample:in CDCl3
qNMR of tablet (0.3495 g tablet, 0.1376 g sample) of "5144 G" extracted with CDCl3 (3×0.7 mL+0.3 mL) + 0.0425 g of 1,4-dimethoxybenzene
11H
1.01.52.02.53.03.54.0 ppm
1.242
1.255
1.259
1.268
1.277
1.286
1.294
1.300
1.307
1.344
1.348
1.357
1.363
1.368
1.441
1.446
1.452
1.458
1.467
1.473
1.477
1.483
1.492
1.497
1.502
1.505
1.511
1.519
1.524
1.528
1.574
1.580
1.582
1.593
1.598
1.611
1.616
1.685
1.693
1.699
1.713
1.722
1.727
1.733
1.742
1.746
1.756
1.760
1.764
1.795
1.800
1.816
2.207
2.226
2.234
2.253
2.501
2.509
2.528
2.536
3.771
3.914
3.929
4.227
4.242
1.1
524
3.0
425
1.1
382
2.9
977
5.3
300
3.4
470
1.2
407
4.5
688
5.1
689
3.1
971
1.0
355
1.0
216
1.0
006
18.9
123
0.9
948
1.0
000
1,4
-dim
eth
oxybenzene
10
5
1
4
O
3
9
8
6
7
11
12
14
13
15
16
17
O
19
1820
OH
H H
H
H
Oxandrolone
Expansion
Oxandro
lone C
H2
-1a
Oxandro
lone C
H2-1
b
Oxandro
lone C
H2-4
a
Oxandro
lone C
H2-4
b
qNMRSample:in CDCl3
qNMR of tablet (0.3495 g tablet, 0.1376 g sample) of "5144 G" extracted with CDCl3 (3×0.7 mL+0.3 mL) + 0.0425 g of 1,4-dimethoxybenzene
11H
210 200 190 180 170 160 150 140 130 120 110 100 90 80 70 60 50 40 30 20 10 0 ppm
-0.000
10.180
13.913
21.007
23.248
25.816
27.150
30.773
31.257
33.816
34.736
35.658
38.742
40.486
45.358
49.706
50.251
55.766
81.036
81.603
114.669
153.781
170.512
Current Data ParametersNAME Sample-5144-G-DiMeOBn_2020-03-16_CDCl3EXPNO 14
PROCNO 1
F2 - Acquisition Parameters
Date_ 20200316Time 23.40INSTRUM spect
PROBHD 5 mm PABBO BB-PULPROG zgpg30TD 65536
SOLVENT CDCl3NS 10600DS 4
SWH 41666.668 HzFIDRES 0.635783 HzAQ 0.7864320 sec
RG 2050DW 12.000 usecDE 7.71 usec
TE 298.7 KD1 2.00000000 secD11 0.03000000 sec
TD0 1
======== CHANNEL f1 ========
SFO1 176.0537397 MHzNUC1 13CP1 9.57 usec
PLW1 60.00000000 W
======== CHANNEL f2 ========
SFO2 700.0828003 MHzNUC2 1HCPDPRG[2 waltz16
PCPD2 65.00 usecPLW2 26.00000000 WPLW12 1.47850001 W
PLW13 0.62465000 W
F2 - Processing parameters
SI 131072SF 176.0352602 MHzWDW EM
SSB 0LB 1.00 HzGB 0
PC 1.40
CDCl3
TMS
10
5
1
4
O
3
9
8
6
7
11
12
14
13
15
16
17
O
19
1820
OH
H H
H
H
Oxandrolone
1,4
-dim
eth
oxybenzene
1,4
-dim
eth
oxybenzene
1,4
-dim
eth
oxybenzene
qNMR sampleSample:in CDCl3
qNMR of tablet (0.3495 g tablet, 0.1376 g sample) of "5144 G" extracted with CDCl3 (3×0.7 mL+0.3 mL) + 0.0425 g of 1,4-dimethoxybenzene
13C{
1H}
10
5
1
4
O
3
9
8
6
7
11
12
14
13
15
16
17
O
19
1820
OH
H H
H
H
O
O
OH
H H
H
H
10
5
1
4
O
3
9
8
6
7
11
12
14
13
15
16
17
O
19
1820
OH
H H
H
H
0.87
1.01
170.6 40.5
33.8
81.6
34.7
45.4
25.8
50.2
35.6
49.7
13.9
10.2
38.7
23.2
31.2
21.0
30.8
27.1
1.491.20
1.190.81
1.46
1.21
1.821.74
1.750.91
1.521.29
1.591.28
1.471.36
4.233.92
2.522.23
1.70
81.0
Sample-5144-G
Oxandrolone
NMR signal assignmentsin CDCl3
+25 °C
Oxandrolone
Numbering scheme
10.0 9.5 9.0 8.5 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 0.0 ppm
0.000
0.792
0.798
0.808
0.809
0.814
0.816
0.825
0.831
0.901
0.907
0.917
0.923
1.167
1.175
1.178
1.185
1.188
1.194
1.200
1.225
1.241
1.250
1.254
1.259
1.268
1.276
1.285
1.289
1.294
1.300
1.302
1.307
1.344
1.348
1.357
1.363
1.367
1.372
1.436
1.440
1.446
1.451
1.458
1.464
1.468
1.473
1.477
1.483
1.488
1.492
1.497
1.500
1.503
1.506
1.512
1.520
1.524
1.530
1.582
1.583
1.586
1.591
1.597
1.599
1.604
1.679
1.684
1.689
1.694
1.697
1.702
1.707
1.710
1.715
1.719
1.724
1.730
1.733
1.735
1.739
1.744
1.753
1.759
1.764
1.796
1.801
1.814
1.817
2.206
2.225
2.233
2.252
2.501
2.510
2.528
2.536
3.916
3.931
4.226
4.242
7.274
1.0
762
2.9
780
1.1
284
2.9
686
5.0
911
2.5
542
1.2
170
4.4
174
1.2
939
3.5
886
1.1
201
1.0
145
1.0
090
1.0
000
1.0
011
Current Data ParametersNAME Sample-5144-G_2020-01-31_CDCl3_28EXPNO 1
PROCNO 1
F2 - Acquisition Parameters
Date_ 20200131Time 21.49INSTRUM spect
PROBHD 5 mm PABBO BB-PULPROG zg30TD 65536
SOLVENT CDCl3NS 80DS 2
SWH 12626.263 HzFIDRES 0.192661 HzAQ 2.5952256 sec
RG 101DW 39.600 usecDE 6.50 usec
TE 298.1 KD1 2.00000000 secTD0 1
======== CHANNEL f1 ========SFO1 700.0847255 MHz
NUC1 1HP1 15.50 usecPLW1 26.00000000 W
F2 - Processing parametersSI 262144
SF 700.0800216 MHzWDW EMSSB 0
LB 0.10 HzGB 0PC 1.00
Qualitative analysis
+25 °C
Sample: 5144 G, extract from tablet 1 in CDCl3
TMS
CHCl3
10
5
1
4
O
3
9
8
6
7
11
12
14
13
15
16
17
O
19
1820
OH
H H
H
H
Oxandrolone
11H
3.54.04.55.05.56.06.57.07.58.08.59.09.510.0 ppm
3.916
3.931
4.226
4.242
7.274
1.0
000
1.0
011
Qualitative analysis
+25 °C
Sample: 5144 G, extract from tablet 1 in CDCl3
CHCl3
10
5
1
4
O
3
9
8
6
7
11
12
14
13
15
16
17
O
19
1820
OH
H H
H
H
Oxandrolone
11H
Expansion
Oxandro
lone
CH
2-1
a
Oxandro
lone
CH
2-1
b
0.80.91.01.11.21.31.41.51.61.71.81.92.02.12.22.32.42.52.6 ppm
0.792
0.798
0.808
0.809
0.814
0.816
0.825
0.831
0.901
0.907
0.917
0.923
1.167
1.175
1.178
1.185
1.188
1.194
1.200
1.225
1.231
1.241
1.250
1.254
1.259
1.268
1.276
1.285
1.289
1.294
1.300
1.302
1.307
1.344
1.348
1.354
1.357
1.363
1.367
1.372
1.436
1.440
1.446
1.451
1.458
1.464
1.468
1.473
1.477
1.483
1.488
1.492
1.497
1.500
1.503
1.506
1.512
1.520
1.524
1.530
1.573
1.582
1.583
1.586
1.591
1.597
1.599
1.604
1.610
1.615
1.676
1.679
1.684
1.689
1.694
1.697
1.702
1.707
1.710
1.715
1.719
1.724
1.730
1.733
1.735
1.739
1.744
1.753
1.759
1.764
1.796
1.801
1.814
1.817
2.206
2.225
2.233
2.252
2.501
2.510
2.528
2.536
1.0
762
2.9
780
1.1
284
2.9
686
5.0
911
2.5
542
1.2
170
4.4
174
1.2
939
3.5
886
1.1
201
1.0
145
1.0
090
Qualitative analysis
+25 °C
Sample: 5144 G, extract from tablet 1 in CDCl3
10
5
1
4
O
3
9
8
6
7
11
12
14
13
15
16
17
O
19
1820
OH
H H
H
H
Oxandrolone
11H
Expansion
Oxandro
lone
CH
2-4
a
Oxandro
lone
CH
2-4
b
Oxandro
lon
e
CH
2-1
6a
Oxandro
lone
CH
2-1
6b
Oxandro
lone C
H2-1
5a
Oxandro
lone
CH
2-1
5b
Oxandro
lone
C
H2-1
2a
Oxand
rolo
ne
CH
2-1
2b
Oxandro
lone
CH
2-7
a
Oxandro
lone C
H2-7
b
Oxandro
lone
CH
2-6
a
Oxandro
lone
CH
2-6
b
Oxandro
lone
CH
2-1
1a
Oxandro
lone
C
H2-1
1b
Oxandro
lone C
H-5
Oxandro
lone
CH
-9
Oxandro
lone
CH
-8
Oxandro
lone C
H-1
4
Oxandro
lone
CH
3-1
8
Oxandro
lone
CH
3-1
9
Oxandro
lone
CH
3-2
0
210 200 190 180 170 160 150 140 130 120 110 100 90 80 70 60 50 40 30 20 10 0 ppm
0.001
10.174
13.916
21.001
23.242
25.808
27.138
30.760
31.248
33.806
34.726
35.647
38.715
40.462
45.353
49.686
50.238
76.868
77.050
77.232
81.038
81.585
170.553
Current Data ParametersNAME Sample-5144-G_2020-01-31_CDCl3EXPNO 3
PROCNO 1
F2 - Acquisition Parameters
Date_ 20200131Time 22.01INSTRUM spect
PROBHD 5 mm PABBO BB-PULPROG zgpg30TD 65536
SOLVENT CDCl3NS 256DS 4
SWH 41666.668 HzFIDRES 0.635783 HzAQ 0.7864320 sec
RG 2050DW 12.000 usecDE 7.71 usec
TE 298.2 KD1 2.00000000 secD11 0.03000000 sec
TD0 1
======== CHANNEL f1 ========
SFO1 176.0537397 MHzNUC1 13CP1 9.57 usec
PLW1 60.00000000 W
======== CHANNEL f2 ========
SFO2 700.0828003 MHzNUC2 1HCPDPRG[2 waltz16
PCPD2 65.00 usecPLW2 26.00000000 WPLW12 1.47850001 W
PLW13 0.62465000 W
F2 - Processing parameters
SI 262144SF 176.0352601 MHzWDW EM
SSB 0LB 1.00 HzGB 0
PC 1.40
210 200 190 180 170 160 150 140 130 120 110 100 90 80 70 60 50 40 30 20 10 0 ppm
Qualitative analysis
+25 °C
Sample: 5144 G, extract from tablet 1 in CDCl3
APT
13C{
1H}
C,CH2 ↑
↓ CH,CH3 CDCl3
TMS
10
5
1
4
O
3
9
8
6
7
11
12
14
13
15
16
17
O
19
1820
OH
H H
H
H
Oxandrolone
95100105110115120125130135140145150155160165170175180185190195200205210215 ppm
170.553
95100105110115120125130135140145150155160165170175180185190195200205210215 ppm
Qualitative analysis
+25 °C
Sample: 5144 G, extract from tablet 1 in CDCl3
APT
13C{
1H}
C,CH2 ↑
↓ CH,CH3
Expansion
10
5
1
4
O
3
9
8
6
7
11
12
14
13
15
16
17
O
19
1820
OH
H H
H
H
Oxandrolone
Oxandro
lone
C-3
10152025303540455055606570758085 ppm
10.174
13.916
21.001
23.242
25.808
27.138
30.760
31.248
33.806
34.726
35.647
38.715
40.462
45.353
49.686
50.238
76.868
77.050
77.232
81.038
81.585
10152025303540455055606570758085 ppm
Qualitative analysis
+25 °C
Sample: 5144 G, extract from tablet 1 in CDCl3
APT
13C{
1H}
C,CH2 ↑
↓ CH,CH3
Expansion
CDCl3
10
5
1
4
O
3
9
8
6
7
11
12
14
13
15
16
17
O
19
1820
OH
H H
H
H
Oxandrolone
Oxandro
lone
C-1
7
Oxandro
lone
CH
2-1
Oxandro
lone
CH
-14
Oxandro
lone
CH
-9
Oxandro
lone
C-1
3
Oxandro
lone
CH
-5
Oxandro
lone
CH
-8
Oxandro
lone
C-1
0
Oxandro
lone
CH
2-1
6
Oxandro
lone
CH
2-4
Oxandro
lone
CH
2-1
2
Oxandro
lone
CH
2-7
Oxandro
lone
CH
2-6
Oxandro
lone
CH
3-2
0
Oxandro
lone
CH
3-1
8
Oxandro
lone
CH
3-1
9
Oxandro
lone
CH
2-1
5
Oxandro
lone
CH
2-1
1
10152025303540455055606570758085 ppm
10.174
13.916
21.001
23.242
25.808
27.138
30.760
31.248
33.806
34.726
35.647
38.715
40.462
45.353
49.686
50.238
76.868
77.050
77.232
81.038
81.585
10152025303540455055606570758085 ppm
Qualitative analysis
+25 °C
Sample: 5144 G, extract from tablet 1 in CDCl3
DEPT^135
CH,CH3 ↑ CH2
↓
13C{
1H}
Expansion
CDCl3
10
5
1
4
O
3
9
8
6
7
11
12
14
13
15
16
17
O
19
1820
OH
H H
H
H
Oxandrolone
Oxandro
lone
C-1
7
Oxandro
lone
CH
2-1
Oxandro
lone
CH
-14
Oxandro
lone
CH
-9
Oxandro
lone
C-1
3
Oxandro
lone
CH
-5
Oxandro
lone
CH
-8
Oxandro
lone
C-1
0
Oxandro
lone
CH
2-1
6
Oxandro
lone
CH
2-4
Oxandro
lone
CH
2-1
2
Oxandro
lone
CH
2-7
Oxandro
lone
CH
2-6
Oxandro
lone
CH
3-2
0
Oxandro
lone
CH
3-1
8
Oxandro
lone
CH
3-1
9
Oxandro
lone
CH
2-1
5
Oxandro
lone
CH
2-1
1