Test report Number 5144 G Object of analysis: 5144 G€¦ · Remarks: In this sample the steroid component was determined to be Oxandrolone. The other components in the tablets were

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


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


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



PROBHD 5 mm PABBO BB-PULPROG zgpg30TD 65536




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


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



PROBHD 5 mm PABBO BB-PULPROG zg30TD 65536




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


+25 °C


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


+25 °C


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



PROBHD 5 mm PABBO BB-PULPROG zgpg30TD 65536




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


+25 °C


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


+25 °C


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


+25 °C


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


+25 °C


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

Documents

Test report Number 5144 G Object of analysis: 5144 G€¦ · Remarks: In this sample the steroid component was determined to be Oxandrolone. The other components in the tablets were