ORGANIC IMPURITIES TESTING OF TENOFOVIR DISOPROXIL

FUMARATE (TDF) TABLETS

Group VI
Dri Waskitho
Debby Citra Dewi
Ni Luh Witariani

Jakarta, 7-11th August 2017

TUJUAN PELATIHAN

 Meningkatkan kompetensi penguji dalam pengujian impuriti

 Mengetahui dan memahami metode pengujian "Impurities
Testing of Tenofovir disoproxil fumarat (TDF) Tablet"
berdasarkan USP Pending Monograph Version 1.
 BACKGROUND
 HIV/AIDS

Source : http://www.who.int/hiv/data/en/, July 2017

BACKGROUND
IUPAC Name :
Bis{[(isopropoxycarbonyl)oxy]methyl} ({[(2R)-1-(6-amino-9H-
purin-9-yl)-2-propanyl]oxy}methyl)phosphonate

SYNONYM : Tenofovir disoproxil fumarate

FORMULA : C19H30N5O10P

Tenofovir Disoproxil

IUPAC Name :
({[(2R)-1-(6-amino-9H-purin-9-yl)propan-2-yl]oxy}methyl)phos
phonic acid

SYNONYM : 9-(2-Phosphonyl-methoxypropyly)adenine (PMPA)

FORMULA : C9H14N5O4P

Tenofovir
PENDAHULUAN
APAKAH TENOFOVIR DISOPROXIL FUMARATE?
 Obat untuk pengobatan infeksi HIV pada orang dewasa dan anak-anak berusia 2 tahun ke atas.
Tenofovir DF selalu digunakan dalam kombinasi dengan obat HIV lainnya.

 Tenofovir DF termasuk dalam kelompok (kelompok) obat HIV yang disebut nucleoside reverse
transcriptase inhibitor (NRTI). NRTI memblokir enzim HIV yang disebut reverse transcriptase. (Enzim
adalah protein yang memulai atau meningkatkan kecepatan reaksi kimia.) Dengan menghalangi
reverse transcriptase, NRTI mencegah HIV berkembang biak dan dapat mengurangi jumlah HIV di
dalam tubuh.
BACKGROUND

 Obat HIV tidak dapat menyembuhkan HIV / AIDS, namun

menggunakan kombinasi obat HIV (disebut rejimen HIV) setiap hari
membantu orang dengan HIV hidup lebih lama, hidup lebih sehat.

 Obat HIV juga mengurangi risiko penularan HIV. Jika Anda memakai
obat-obatan HIV, termasuk tenofovir DF, jangan mengurangi,
melompati, atau berhenti meminumnya kecuali jika petugas
kesehatan Anda memberi tahu Anda.
(https://aidsinfo.nih.gov/drugs/290/tenofovir-disoproxil-
fumarate/0/patient)
BACKGROUND

 Impurities
USP 40-NF 35 General Chapter <1086>
Impurities in Drug Substance and Drug Product
Impurity :
“Any component of a drug substance that is not the chemical entity
defined as the drug substance and in addition, for a drug product, is any
component that is not a formulation ingredient”
BACKGROUND

 Organic Impurities can arise during manufacturing process and/or

storege of the drug substances, include:
 Starting Material
 By Products
 Intermediate
 Degradation Product
 Reagents, Ligands and catalysts
 Geometric and Stereoisomers
Impurities of Tenofovir Disoproxil Fumarate (TDF) Tablet

 Process impurities, synthethic by product, residual solvents, heavy

metals, and other inorganic and organic impurities may be present in the
drug substances and excipients used in the manufacture of the drug
product and should be assessed and controlled.
Source : USP General Chapter <3>Topical and Transdermal
Drug Products-Product Quality Tests, USP 40-NF 35

 Because some impurities might have toxicity

 Because some impurities might have
Method Guidance

ASSAY TESTING IMPURITIES TESTING

Chromatography ; System VS
Suaitability
<621>
General notices (5.50)
Validation of compendial
procedures <1225>
Chromatography ; System
Suaitability
<621>
General notices (5.60)
Impurities in drug
substances and drug
products<1086>
Validation of compendial
procedures <1225>
Equipment And Materials
Equipment

Name Identity No. Brand/Type Cal. Due Date

High Performance Liquid 10/LC/PT Shimadzu Prominence 26 Oct 2017
Chromatograph LC 20AD
Balance 09/BL/PT Sartorius ME 5F 28 Dec 2018
pH Meter 03/PH/PT 03 Jan 2018
Sonicator 07/UL/PT Branson 550 N/A
Water Purification System 03/WP/PT Milli Q – Q-POD N/A
Column
Equipment And Materials
Reagents
Name Lot No Expiry Date
Disodium Hydrogen Phosphate (Merck) K4715198603 30 Sep 2020
Phosphoric Acid (Merck)
Methanol (Merck) I837707626 30 Jun 2019
tert-Butyl Alcohol (Merck)
Purified Water

Reference standards
Name Lot No Purity Loss on Drying
Tenofovir Disoproxil Fumarate USPRS GDM335 0.999 mg/mg -
Tenofovir USPRS R044CD -
Adenine USPRS IDM090 -
Equipment And Materials
Sample Information
Sample Name : Tenofovir Disoproxil Fumarate
Dosage Form : Tablet
Composition : Tenofovir Disoproxil Fumarate 300 mg
Appearance : Light blue, round, biconvex tablets, imprinted with “M”
on one side and “153” on other side
Reg. No. : GKX1340400217A1
Package : Bottle @ 30 tablets
Manufacturer : Mylan Laboratories Ltd.
Importer/Distributor : PT. Kimia Farma Tbk., Jakarta-Indonesia
Batch No. : 3061232
Mfg./Expiry Date : Nov 2016 / Oct 2019
 Mobile Phase Preparation
2
1 0.01 M Na2HPO4 Buffer Solution (pH 5.5)
Solution A
Methanol-tertiary butanol-
buffer (11:1:28)
For 1000 mL:
8.9 g Na2HPO4 Methanol = 275 mL
Water Tertiary butanol = 25 mL
0.01 M Na2HPO4
Buffer = 700 mL
5000 mL 5000 mL 5000 mL

a)
About 8.9 g of Na2HPO4
is weighed on an
analytical balance and
transferred into a 5000
mL volumetric flask
Figure 1. Preparation of mobile phase
b)
A portion of water is
c)
The mixture is
d)
Additional water is added
3
added to the volumetric swirled until all of up to the mark on the Solution B
flask the solid has volumetric flask and the Methanol-tertiary butanol-
dissolved solution is adjusted to a buffer (27:1:12)
completely pH of 5.5 with phosphoric
acid For 1000 mL:
Methanol = 675 mL
Tertiary butanol = 25 mL
Buffer = 300 mL
System Suitability Solution

1 2

+
up to 50 mL

3.75 mg a)
5.0 mL of adenine and
tenofovir stock solution is
each measured using
volumetric pipette
b) c)
The measured volume The measured volume in the
of stock solution is second flask is then diluted
transferred into 50 mL with solution A up to the
volumetric flask volumetric mark

3 5.0 mL of solution (2) is finally diluted with solution

Figure 2. SST A in 50 mL volumetric flask up to the volumetric
mark (0.75 µg/mL of tenofovir and adenine)
 Standard Solution

1 2

+
up to 100 mL

5 mg of tenofovir
disoproxil fumarate
a)
5.0 mL of tenofovir disoproxil
fumarate stock solution is
measured using volumetric
pipette
b) c)
The measured volume The measured volume in the
of stock solution is second flask is then diluted
transferred into 50 mL with solution A up to the
volumetric flask volumetric mark

3 5.0 mL of solution (2) is finally diluted with solution

Figure 3. Std A in 50 mL volumetric flask up to the volumetric
mark (0,5 µg/mL of tenofovir disoproxil fumarate)
 Sample Solution

1 2

a)
NLT 20 tablets equivalent to 6000 mg/mL
tenofovir disoproxil fumarate is add with
about 3500 mL of solution A in 5000 mL
volumetric flask

b)
The mixture is sonicated for 10 minute,
cooled to room temperature, and then

mark 3
diluted with solution A up to the volumetric
a) b) c)
4.0 mL of sample stock solution The measured volume The measured volume in the
is measured using volumetric of stock solution is second flask is then diluted
pipette transferred into 25 mL with solution A up to the

Figure 4. Preparation of volumetric flask volumetric mark

sample solution
Sample solution: 500 µg/mL
Chromatographic Conditions
Instrument: HPLC Shimadzu Prominence LC 20AD
Chromatographic system:
Table 1 Table 2
Detector 260 nm Time (min) % Solution A % Solution B
Column XBridge C18; 4,6 x 25 cm; 5 0.00 – 2.00 100 0
µm 2.01 – 30.0 0 100
Lot No. 0173352721 (Code
067) 30.01 – 45.00 100 0
Column 35 °C 45.01 – 65 100 0
temperature
Table 3
Sample 4 °C (freshly taken out from
temperature refrigerator prior to Parameters Requirements
injection) Tailing factor NMT 2,0
Flow rate 1.0 mL/min Resolution NLT 1.5 (tenofovir-
Injection volume 10 µL adenine)
Gradient system See Table 2 RSD NMT 10.0%
System Suitability
Table 3
Parameters Requirements Samples
Tailing factor NMT 2,0 Standard solution
Resolution NLT 1.5 (tenofovir-adenine) System suitability solution
RSD NMT 10.0% Standard solution

Bracketing

% Diff Requirement: NMT 2.0%

Workplan

Table 4. Injection sequence

No Solution No Solution
1 Solvent/blank 9 Control standard solution
2 SST solution 10 Control standard solution
3 Standard solution 11 Sample solution 1
4 Standard solution 12 Sample solution 2
5 Standard solution 13 Sample solution 3
6 Standard solution 14 Sample solution 4
7 Standard solution 15 Standard solution
8 Standard solution 16 SST solution
 Data Interpretation
Table 4.
Table 5. Injection sequence
Acceptance Criteria

Organic impurities testing of TDF tablet <USP Pending monograph v1>

Results
Solvent/Blank
mV
Detector A:260nm
1.5

1.0
mV

0.5

0.0

0.0 5.0 10.0 15.0 20.0 25.0 30.0 35.0 40.0 45.0 50.0 55.0 60.0 min

Figure 5. Chromatogram obtained from blank solution (solvent)

Results
System Suitability Solution (Resolution)
mV

Adenine/2.880
Tenofovir/2.609
Detector A:260nm

5.0

2.5

0.0
0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 5.0 5.5 min

Figure 6. Chromatogram obtained from SST solution

Resolution
No Name RT (min) Area TF Conclusion
Result Criteria
1 Tenofovir 2.609 23867 1.290 -- -- --
2 Adenine 2.880 40916 1.373 1.638 NLT 1.5 Passed
Results
Standard Solution
mV
Detector A:260nm
3

TDF/19.827
Fumaric Acid/2.378

0.0 5.0 10.0 15.0 20.0 25.0 30.0 35.0 40.0 45.0 50.0 55.0 60.0 min

Figure 7. Chromatogram obtained from Std solution

No Name RT (min) Area TF Resolution Theor. Plate
1 Fumaric Acid 2.378 512 1.225 -- 4250.450
2 TDF 19.827 7329 1.175 70.299 51184.754
Results
System Suitability Test
Parameters Criteria Results Conclusion
Tailing factor NMT 2.0 1.175 Passed
Resolution NLT 1.5 1.638 Passed
RSD NMT 10.0% 1.9656 Passed

Quality Assurance
Working Std Control Std
 Results Sample Analysis
mV

RT2.382/2.382
RT4.090/4.090

TDF/19.787
Detector A:260nm

RT35.165/35.165
RT28.598/28.598
RT25.312/25.312
1.5

1.0

0.5

0.0
0.0 5.0 10.0 15.0 20.0 25.0 30.0 35.0 40.0 45.0 50.0 55.0 60.0 min

Figure 8. Chromatogram obtained from Sample solution

Sample 1
Acceptance
Tailing Theoretical C Spl C Std 0,05% % each
No Name RT Area Resolution RRT F Keterangan criteria
factor Plate (ug/mL) (ug/mL) main peak impurity
NMT (%)
1 RT2.382 2.382 434958 1.335 -- 3.685.213 0.120 480 0,56462 3046,42 fumaric acida -
tenofovir isoproxil
2 RT4.090 4.090 58327 1.230 9.097 5.602.809 0.207 480 0,56462 1,5 3046,42 0,634099 3,0
monoester
3 TDF 19.787 6092840 1.218 54.449 48.874.895 0.999 480 0,56462 3046,42 TDF
4 RT25.312 25.312 1653 1.211 16.978 119.574.396 1.278 480 0,56462 3046,42 disregard
5 RT28.598 28.598 2394 1.208 10.780 130.447.209 1.444 480 0,56462 3046,42 disregard
tenofovir
6 RT35.164 35.164 15963 0.775 15.461 69.703.466 1.776 480 0,56462 1 3046,42 0,260311 0,75
disoproxil dimer
Total impurities 0,89441 4,0
 Results
Sample 2
Acceptance
Tailing Theoretical C Spl C Std 0,05% % each
No Name RT Area Resolution RRT F Keterangan criteria
factor Plate (ug/mL) (ug/mL) main peak impurity
NMT (%)
1 RT2.381 2.381 435596 1.333 -- 3.739.092 0.120 480 0,56462 3046,42 fumaric acida -
tenofovir isoproxil
2 RT4.089 4.089 62273 1.227 9.127 5.594.383 0.207 480 0,56462 1,5 3046,42 0,676997 3,0
monoester
3 TDF 19.790 6106907 1.219 54.425 48.823.831 0.999 480 0,56462 3046,42 TDF
4 RT25.307 25.307 1731 1.247 16.719 112.539.811 1.278 480 0,56462 3046,42 disregard
5 RT28.597 28.597 2433 1.232 10.667 131.823.658 1.444 480 0,56462 3046,42 disregard
tenofovir
6 RT35.165 35.165 17862 0.781 15.068 63.828.131 1.776 480 0,56462 1 3046,42 0,291279 0,75
disoproxil dimer
Total impurities 0,968276 4,0

Sample 3
Acceptance
Tailing Theoretical C Spl C Std 0,05% % each
No Name RT Area Resolution RRT F Keterangan criteria
factor Plate (ug/mL) (ug/mL) main peak impurity
NMT (%)
1 RT2.381 2.381 445200 1.339 -- 3.695.295 0.120 480 0,56462 3046,42 fumaric acida -
tenofovir isoproxil
2 RT4.091 4.091 70828 1.232 9.096 5.567.396 0.207 480 0,56462 1,5 3046,42 0,770002 3,0
monoester
3 TDF 19.798 6238063 1.222 54.341 48.713.952 1.000 480 0,56462 3046,42 TDF
4 RT25.317 25.317 1751 1.181 16.947 119.837.778 1.279 480 0,56462 3046,42 disregard
5 RT28.608 28.608 2566 1.173 10.762 128.614.602 1.445 480 0,56462 3046,42 disregard
tenofovir
6 RT35.172 35.172 17542 0.766 15.181 66.482.002 1.776 480 0,56462 1 3046,42 0,28606 0,75
disoproxil dimer
Total impurities 1,056063 4,0
Results
Sample Analysis
% Acceptance
RRT
No. Peak Name F RT Area RT Impurit Crit. Conclusion
refer.
y NMT (%)
1 Fumaric acid 0.12 - 2.381 438585 0.120 - - -
2 Tenofovir isoproxil
0.14 1.500 4.090 63809 0.207 0.6937 3.0 Meets req.
monoester
3 Tenofovir disoproxil 1.0 - 19.792 6145937 0.999 - - -
4 Disregard peak - - 25.312 1712 1.278 - - -
5 Disregard peak - - 28.601 2464 1.444 - - -
6 Tenofovir disoproxil
1.74 1.000 35.167 17122 1.776 0.2792 0.75 Meets req.
dimer
Total impurities -- -- -- -- -- 0.9729 4.0 Meets req.
Discussion
Results
Sample Analysis
% Acceptance
RRT
No. Peak Name F RT Area RT Impurit Crit. Conclusion
refer.

Thank You
y NMT (%)
1 Fumaric acid 0.12 - 2.381 438585 0.120 - - -
2 Tenofovir isoproxil
0.14 1.500 4.090 63809 0.207 0.6937 3.0 Meets req.
monoester
3 Tenofovir disoproxil 1.0 - 19.792 6145937 0.999 - - -
4 Disregard peak - - 25.312 1712 1.278 - - -
5 Disregard peak - - 28.601 2464 1.444 - - -
6 Tenofovir disoproxil
1.74 1.000 35.167 17122 1.776 0.2792 0.75 Meets req.
dimer
Total impurities -- -- -- -- -- 0.9729 4.0 Meets req.
Conclusion
 Tablet Tenofovir Disoproxil Fumarate : meets
requirement for impurities
Pay attention to :
(GLP Implementation)
1. Develop workplan
2. Protect yourself with the personal protection stuff (APD)
3. Make sure all the instrument are calibrated and in a good condition
4. Make sure all the reagent is correct and not expired
5. Documenting every process and result in a good documentation
practise