3. QbD: A systematic approach to development that begins with predefined objectives and
emphasizes product & process understanding and process control, based on sound science and
quality risk management.
•Submission of a design space to FDA is a pathway obtaining the ability to operate within that
design space without further regulatory approval
•Relevant documents from the ICH, ICH Q8 Pharmaceutical Development, along with ICH Q9
Quality Risk Management and ICH Q10 Pharmaceutical Quality Systems, indicate on an
abstract level how quality by design acts to ensure drug product quality.
INTRODUCTION
UPLC Advantages over HPLC
•More information within short time
•Better situational response time (Take the research decisions within short time with
more information, process monitoring ,product release )
•More robust method development
Chapter-1
DEVELOPMENT AND VALIDATION OF DULOXETINE.HCl
RS METHOD BY UPLC USING QbD
3
4. DRUG PROFILE
Duloxetine.HCl structure
Chemical formula C18H19NOS. HCl
Molecular weight 333.38
IUPAC name (+)–(S)-N-methyl-γ-(1-
naphthyloxy)-2-thiophene
propylamine hydrochloride.
Solubility Freely soluble in methanol,
ethanol, chloroform, & di
methyl sulfoxide.
Soluble in acetonitrile,
acetone.
pka 9.5
λmax 230 nm
Melting point 140-143 oC
.HCl
4
5. Mechanism of action
Medical uses
•Major Depressive disorder
•Stress urinary incontinence
•Diabetic peripheral neuropathy
•Generalized anxiety disorder
•Fibromyalgia
•Chronic fatigue syndrome
•Interstitial cystitis
MOA : Duloxetine Serotonin Norepinephrine Reuptake Inhibitors- block proteins in the pre-synaptic
neuron that act as re-uptakers. This increases the concentration of both neurotransmitters at the synaptic cleft.
5
6. S.
no.
Title of the article Column Mobile phase composition
(%V/V)
Flow
rate
(ml/min)
Wave
Length
(nm)
Refer
ences
1 Development and validation of Stability
indicating RP HPLC method for duloxetine.HCl
in its bulk and dosage forms.
Hypersil C-18 (250mm
X 4.6 mm id, 5μm )
Acetonitrile:0.01M potassium dihydrogen phosphate
buffer (pH 5.4 adjusted with ortho phosphoricacid )
(50:50)(%v/v)
1.0
ml/min
229nm 31
2 Analysis of Duloxetine Hydrochloride and Its
Related Compounds in Pharmaceutical Dosage
Forms and In Vitro Dissolution Studies by
Stability Indicating UPLC
UPLC C-18
(50 mm x 4.6mm,1.8
μm)
Mobile phase A : 0.01M potassium di hydrogen
phosphate (pH 4.0) buffer, tetrahydro furan, and
methanol (67:23:10 ).
Mobile phase B : 0.01 M potassium dihydrogen
phosphate, (pH 4.0) buffer, Acetonitrile 60:40 (v/v).
0.6
ml/min
236nm 32
3 Development and validation of UPLC method
for determination of Duloxetine.HCl residues
on pharmaceutical mfg equipment surfaces .
UPLC HSS T3
(100 x 2.1mm,1.7 μm)
0.01Mpotassiumdihydrogen phosphate(pH 3.0)
buffer, and Acetonitrile(60:40)
0.4
ml/min
230 33
4 Stability Indicating Nature of RP-HPLC method
for Determination of Impurity profile and
Degradation impurities in Duloxetine
Hydrochloride
YMC Pack C8
(250 X 4.6 mm,
5μm )
Solvent A (0.01 M of Sodium Di hydrogen
Orthophosphate and 1.0g of 1-Heptane Sulfonic Acid
Sodium Salt transfer in 1000 ml of water, pH 3.0
using Orthophosphoric acid) and
Solvent B (Acetonitrile).
1ml/min 217 34
5 Development and Validation of a LC/MS/MS
Method for the Determination of Duloxetine in
Human Plasma and its Application to
Pharmacokinetic Study
X-terra RP8
(50mm x4.6 mm, 5μm )
30mM Ammonium formate (pH-5.0 ) and acetonitrile
as an isocratic
0.4
ml/min
230nm 35
6 A validated RP- HPLC method for the analysis
of duloxetine hydrochloride in pharmaceutical
dosage forms
Inertial BDS C8
(250 x 4.6 mm,5 am)
Buffer: Acetonitrile: Methanol (55:37:8%)
1ml/min 215nm
36
7 Method Development and Validation of
Duloxetine Hydrochloride by RP HPLC
BDS Hypersil C18
(150 x 4.6 mm,5 μm)
Buffer :Mixed phosphate buffer (1.625g
Potassiumdihydrogen phosphate+0.3g Di potassium
hydrogen phosphate in 550ml water)
1ml/min 232nm
37
LITERATURE REVIEW
6
7. AIM AND OBJECTIVE
The objective of the work is to develop a stability indicating RP UPLC LC-MS compatible
related substances method for the determination of Duloxetine. HCl and its related impurities
Present study includes:
Development of stability indicating LC-MS compatible related substances method by
using QbD Fusion AE software (UPLC).
Forced degradation studies according to ICH Guidelines.
Validation of the method according to ICH Guidelines.
7
14. Specificity
Acid degradation with 1N HCl at room temperaure for 3 hrs.
Acid degradation with 1N HCl at 60C temperaure for 3 hrs.Base degradation with 1N NaOH at 60C temperaure for 3 hrs.Peroxide degradation with 5% H202 at 30C temperaure for 3 hrs.
Base degradation with 1N NaOH at 30C temperaure for 3 hrs.Peroxide degradation with 5% H202 at 30C temperaure for 1 hr.
METHOD VALIDATION
14
15. Degradation
Conditions
% Increase Of
Alcoholimpurity
RRT-0.24
% Increase
Of
Impurity C
RRT-0.52
% Increase Of
Naphthol Impurity
RRT-0.81
% Increase Of
Di Methyl
Impurity
RRT-1.53
Purity
Angle
Purity
Threshold
Purity
Test
Acid deg. at RT for
3 hrs
3.93 4.06 16.84 0.01 0.187 1.024 Pass
Acid deg.at 60°C for
1 hr
4.33 2.72 18.31 0.01 0.083 0.738 Pass
Base deg.at RT for
3 hrs
- 0.15 - - 0.087 0.592 Pass
Base deg. at 60°C
for 3 hrs
3.42 0.40 1.59 0.01 0.061 0.443 Pass
Peroxide deg. at RT
for 1 hr
0.09 0.13 0.12 0.01 1.371 5.182 Pass
Peroxide deg. at RT
for 3 hr
0.14 0.18 0.08 0.01 0.795 5.366 Pass
Heat deg. at 60°C
for 3 hrs
0.02 - 0.03 0.01 0.092 0.461 Pass
Heat deg. at 60°C
for 24 hrs
- 0.24 4.05 - 0.065 0.554 Pass
UV solution
stability
0.09 0.06 0.12 0.01 0.056 0.305 Pass
Degradation summary
15
16. Degradation summary
Degradation
Conditions
% Increase Of
Alcoholimpurit
y
RRT-0.24
% Increase
Of
Impurity C
RRT-0.52
% Increase Of
Naphthol
Impurity
RRT-0.81
% Increase Of
Di Methyl
Impurity
RRT-1.53
Purity
Angle
Purity
Threshold
Purity
Test
White fluorescence
light at 1.2 milllion LUX hrs for
5days
- 0.01 0.01 0.03 0.123 0.876 Pass
UV at 200Watt hrs/m2
for 5days
- - 0.04 - 0.108 0.479 Pass
Heat Degradation
at 105°c for 5 days
- - - 0.02 0.076 0.321 Pass
Chromatogram of Control Sample (solid) exposed to white fluorescent light for 5daysChromatogram of Control Sample (solid) exposed to UV light for 5 daysChromatogram of Control Sample (solid) exposed to 105°C for 5days
16
18. Specificity-Impurity interference
S.
No
.
Name of
Impurity/Analyte
Rt from
individual
injection
Rt from
impurities
spiked
sample
solution
1.
Alcohol impurity
0.8 0.8
2.
Impurity C
1.8 1.8
3.
Naphthol impurity
2.8 2.8
4.
Duloxetine.HCl
3.5 3.4
5. Dimethyl impurity
5.3 5.3
Chromtogram of reference solution
Chromtogram of 100% spike solution
Chromtogram of blank
18
19. S.No Peak area of Duloxetine.HCl
1 4894
2 4815
3 4758
4 4800
5 4766
6 4701
Average 4877
%RSD 1.4
Theoretical plates of
Duloxetine. HCl
3073
Resolution between Naphthol
imp& Duloxetine HCl peaks
3.5
Peak tailing of Duloxetine
HCl.
1.0
System suitability
Acceptance criteria:
•The resolution between Naphthol imp&
Duloxetine HCl should be not < 2.
•The no. of theoretical plates for Duloxetine peak
should be not < 2000.
•Peak tailing of Duloxetine HCl should be not
more than 2
Conclusion:
•The resolution between Naphthol imp&
Duloxetine HCl was found to be >2
•Plate count for Duloxetine HCl observed to be
>2000
•Peak tailing of duloxetine HCl was found to be
1.0.
19
20. LOD ResultsLOQ Results
S.NO. Name Retention
Time
Area
%
Area
Height
RT
Ratio
USP
Resolution
USP
Tailing
USP Plate
Count
1
ALCOHOL
IMP
0.83 261 5.27 69 0.248 1.1 939.70
2 IMP C 1.30 511 10.31 98 0.387 3.7 1.0 1191.68
3
NAPHTHOL
IMP
2.69 1174 23.68 188 0.798 8.8 1.2 3998.90
4 DXT STD 3.38 621 58.32 448 1.000 3.9 1.1 5584.05
5
DI METHYL
IMP
5.16 120 2.42 15 1.529 8.8 0.9 7529.00
S.
no
.
Name
Retention
Time
Area % Area Height
RT
Ratio
USP
Resolution
USP
Tailing
USP
Plate
Count
1
ALCOHOL
IMP
0.81 643 7.47 138 0.241 1.2 643.49
2 IMP C 1.72 1280 14.86 214 0.508 6.2 1.1 1700.58
3
NAPHTHOL
IMP
2.69 4015 46.61 610 0.794 5.6 1.0 3495.80
4 DXT STD 3.39 2140 24.85 301 1.000 3.7 1.1 4819.91
5
DI METHYL
IMP
5.20 535 6.21 56 1.534 6.2 0.9 1999.08
20
23. Accuracy Results
NAME %Recovery at each level %RSD at each level
Acceptance criteria Other than
LOQ Level
90 to 110%
At LOQ
Level
(70 to 130%)
Other than
LOQ
Level
NMT 5.0
At LOQ
Level
(NMT 5.0)
Alcohol impurity 97.1 to
103.4
113.9 to 117.5 1.59 to
2.79
1.81
Impurity C
97 to 104.5 92.5 to 96.1
0.77 to
2.36
2.22
Naphthol impurity 94.7 to
103.9
106.6 to 110.9
0.53 to
3.42
2.37
Dimethyl impurity 92.7 to
105.6
97.7 to 109.5
1.72 to
1.52
2.62
23
24. Precision at LOQ level
No of
Injections
at 100%
level
Area
Alcohol
impurity
Imp c Napthol
impurity
Di
methyl
impurity
Duloxetine
.HCl
Inj 1 655 1298 4041 576 2078
Inj 2 682 1290 4070 570 2096
Inj 3 655 1297 4008 553 2105
Inj 4 697 1283 4059 532 2197
Inj 5 654 1219 4012 562 2177
Inj 6 643 1280 4015 524 2140
Average 659 1277 4034 556 2135
STDEV 19.00 29.71 26.70 18.02 52.21
%RSD 2.88 2.33 0.66 3.24 2.44
Acceptance Criteria:
The % RSD of peak areas for each
impurity should be≤ 5.0
Conclusion:
The %RSD was found to be within
the limits and results were
satisfactory
PRECISION
No of Injections
at 100% level
Area
Alcohol
impurity
Impurity
C
Naphthhol
impurity
Di methyl
impurity
Duloxetine.
HCl
Inj 1 4876 11569 29247 12651 7034837
Inj 2 4765 11023 29456 12486 7128765
Inj 3 5142 11432 28865 12980 7295432
Inj 4 5121 11675 29087 12789 7238766
Inj 5 4873 11569 29780 12678 7332678
Inj 6 5016 11238 28789 12564 7167468
Average 4965 11786 29204 12691 7199657
STD DEV 151.43 245.06 373.62 174.96 111017.47
%RSD 3.05 2.08 1.28 1.38 1.54
Precision at 150% level
24
25. System Precision
No of
Injections
at 100%
level
Area
Alcohol
impurity
Imp
C
Naphthol
impurity
Di methyl
impurity
Duloxetine .Hcl
Inj 1 3026 7036 18587 4466 3026
Inj 2 3119 7016 18621 4445 3119
Inj 3 3045 7088 18481 4265 3045
Inj 4 3131 7092 18683 4419 3131
Inj 5 3092 7053 18582 4339 3092
Inj 6 3009 7046 18593 4040 3009
Average 3070 7055 18591 4328 3070
STDEV 50.71 79.75 65.53 160.1 50.72
%RSD 1.73 0.41 0.42 3.74 1.71
Acceptance Criteria:
The % RSD of peak areas of
replicate injections for each
impurity should be≤ 5.0
Conclusion:
The %RSD was found to be
within the limits and results were
satisfactory.
25
26. Method Precision
No of
Injections
at 100%
level
Area % Recovery of impurity
Alcohol
impurity
Imp
C
Naphthol
impurity
Di
methyl
impurity
Alcohol
impurity
Imp
C
Naphthol
impurity
Di methyl
impurity
Inj 1 3653 5885 18587 7364 0.15 0.15 0.13 0.13
Inj 2 3671 5598 18621 6991 0.15 0.14 0.13 0.12
Inj 3 3693 5591 18481 6996 0.15 0.14 0.13 0.12
Inj 4 3702 5698 18683 7368 0.15 0.14 0.13 0.12
Inj 5 3663 6075 18582 7184 0.15 0.14 0.13 0.12
Inj 6 3643 6041 18593 6995 0.14 0.15 0.13 0.12
Average 3670 5814 18591 7949 0.15 0.15 0.13 0.12
STDEV 22.88 216.
58
65.57
183.03
0.00 0.00 0.00 0.00
%RSD 0.62 3.73 0.35 2.30 2.75 2.95 0 3.36
Acceptance Criteria:
The % RSD of Recovery obtained
for each impurity should be≤ 5.0
Conclusion:
The %RSD was found to be within
the limits and results were
satisfactory.
26
27. Para
meters
Change of
conditions
USP Plate
Count of
Duloxetine.
HCl
Tailing
factor for
Duloxetine.
HCl
Resolution
between
Naphthol
impurity &
Duloxetine.HCl
Flow
variation
(ml/min)
Actual 1 4442 1.1 3.57
Low 0.9 5084 1.1 3.57
High 1.1 3509 1.2 3.21
Buffer
pH
variation
Actual 6.6 4442 1.1 3.57
Low 6.4 4590 1.1 3.73
High 6.8 4482 1.1 4.34
Column
oven
temp.
variation
(°C)
Actual 40 4442 1.1 3.57
Low 38 4022 1.2 3.32
High 42 4142 1.0 3.36
Robustness
Acceptance criteria:
•The resolution between Naphthol
imp& Duloxetine HCl should be
not < 2.
•The no. of theoretical plates for
Duloxetine. HCl peak should be not
less than < 2000.
•Peak tailing of Duloxetine HCl
should be not more than 2.0
Conclusion:
•The resolution between Naphthol
imp& Duloxetine HCl was found to
be >2
•plate count for Duloxetine HCl
peak observed to be >2000
• peak tailing of duloxetine HCl was
found to be 1.0.
27
28. CONCLUSION
From the results method considered as simple and precise with a shorter run time of
8minutes, which can be used for separation and quantification of related substances
of Duloxetine HCl.
The method developed was validated and the method was inferred to be linear,
accurate precise and robust based on the results of validation.
The method developed was economical as the time required and solvent consumption
for the complete analysis is less.
A Robust method for duloxetine was developed in 2days using QbD approach on an ACQUITY
UPLC H-Class sytem running Empower 2 & Fusion AE Software
28
29. Plasma generates
positive ions
Detector
(e.g. electron multiplier)
Sorted by mass analyser, e.g.
quadrupole, magnetic sector,
according to m/z ratio
Spray chamber
sample
Nebuliser Interface
Under vacuum
Chapter-2
INTRODUCTION
IMPURITY PROFILING OF MILNACIPRAN.HCL BY ICP-MS
29
30. ICP-MS
Advantages
Excellent detection limits for most elements in Periodic Table (low ppb - ppt for
all elements)
Wide dynamic range (8 to 9 orders)
Much simpler spectra than optical techniques
Low sample volume consumption
Mass spec - so isotopic information available
Good sample throughput
Flexible quantitation methods
semiquantitative
external calibrations
isotope ratios
Disadvantages
Dissolved solids/matrix effects
Capital cost high
Requires knowledgeable operator
INORGANIC IMPURITIES
CLASSIFICATION ORAL EXPOSURE PARENTERAL EXPOSURE INHALATION
EXPOSURE*
PDE(µg/day) CONCENTRATION
(ppm)
PDE(µg/day) CONCENTRATION
(ppm)
Class 1 A
Pt,Pd 100 10 10 1 Pt:70
Class 1 B
Ir,Rh,Ru,Os 100** 10** 10** 1**
Class 1 C
Mo,Ni,Cr,V metals of
significant toxicity
250 25 25 2.5 Ni:100
Cr(VI):10
Class 2
Mn,Cu
Metals with low
safety concern
2500 250 250 25
Class 3
Fe,Zn
Metals with minimal
safety concern
13000 1300 1300 130
30
31. System suitability
S.
N
o
Element Standard
conc.
(in ppb)
Check standard
conc.
(in ppb)
%Recovery Correlation
coefficient
1 Aluminium 200 203.722 101.9 0.9999
Acceptance criteria:
Correlation coefficient should be not less than 0.99 for linearity and recovery
value for check standard should be between 80% to 120%.
Inference:
Results obtained were within the limits.
S
.
N
o
.
Element Theoretical
(in ppm)
Measured
(in ppm)
% Recovery
1 Aluminium 100.0 102.4 102.4
Acceptance criteria:
Percentage recovery should be between 70.0% to 150.0% for Aluminium in the
presence of sample and matrix components.
Inference:
Results obtained were within the limits.
Specificity
31
32. LOD,
S. No. Aluminium
(counts per second)
1 35382.72
2 33338.96
3 31872.15
4 32091.21
5 31853.64
6 31659.90
Mean 32699.76
SD 1446.33
%RSD 4.4
Acceptance criteria:
LOD: The response of LOD solution for Aluminium Should be consistently detected.
LOQ: The % RSD of response of LOQ solution for Aluminium should be not more than 20.
Inference: Results obtained were within the limits.
S. No.
Aluminium
(counts per second)
1
13650.27
2
14183.95
3
14233.55
4
14885.10
5
15080.25
6
15329.92
LOQ
LOD LOQ
32
33. Linearity
Conc. in ppb Conc. w.r.to
sample(ppm)
Aluminium
(Counts per
second)
40.0 20 34921.84
100.0 50 83234.79
200.0 100 166588.4
300.0 150 260151.97
400.0 200 341173.87
Correlation
coefficient
--
0.9997
Slope -- 1717.8806
Intercept -- -1445.4129
Acceptance criteria:
The correlation coefficient should not be less than 0.99.
Inference:
Results obtained were within the limits.
33
34. Spike
level
Actual ppm
Measured
ppm
% recovery % RSD
LOQ
20.0 20.1 100.5
1.220.0 20.3 101.5
20.0 20.6 103.0
50%
50.0 46.4 92.8
0.250.0 46.2 92.4
50.0 46.4 92.8
100%
100.0 116.4 116.4
10.3100.0 97.1 97.1
100.0 98.6 98.6
150%
150.0 157.3 104.9
1.8150.0 162.4 108.3
150.0 162.3 108.2
Acceptance criteria:
•The percentage recovery calculated for each
level should be in the range of 70-150.
• The percentage relative standard deviation of the
recoveries obtained for Aluminium should be not
more than 20.
Inference:
Results obtained were within the limits
Accuracy
34
35. System precision
S. No. Aluminium
(Counts per second)
1 150817.50
2 150787.60
3 152015.19
4 153737.09
5 157131.47
6 157633.09
Mean 153686.99
SD 3061.83
%RSD 2.0
Acceptance Criteria:
% RSD for counts per second of Aluminium should be not more than 20.
Inference:
Results obtained were within the limits.
Prep. No. Aluminium
(% Recovery)
1 103.0
2 103.2
3 119.0
4 106.5
5 105.3
6 109.5
Mean 107.8
SD 6.0076
%RSD 5.6
Method precision
Acceptance Criteria:
% RSD for recovery of Aluminium should be not more than 20.
Inference:
Results obtained were within the limits.
35
36. Prep. No.
Aluminium (% Recovery)
Analyst-1/Day-1 Analyst-2/Day-2
1 103.0
91.0
2 103.2
93.3
3 119.0
90.2
4 106.5
91.7
5 105.3
93.0
6 109.5
93.0
Mean 107.8
92.0
SD 6.0076
1.266
%RSD 5.6
1.4
Acceptance Criteria:
% RSD for recovery of Aluminium for
individual analyst should be not more
than 20.
Inference:
Results obtained were within the limits.
Intermediate precision
36
37. CONCLUSION
A Novel ICP-MS method was developed and validated for the determination of
Aluminium content in Milnacipran.
Linearity was conducted from 20% to 200% with respect to sample solution,
correlation coefficient was found to be 0.999. LOD, LOQ were found to be
6.3ppm, 20ppm respectively.
Statistical analysis proved the method is repeatable, specific, simple, rapid,
precise, accurate for the estimation Aluminium content in Milnacipran
37
38. REFERENCES
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23. http://www.sigmaaldrich.com/etc/medialib/docs/Aldrich/General_Information/lc_ms_brochure.
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29. Sean C Sweet Man.; Fr Phams. Matrindale.; The Complete Drug Reference,2007,5th Edition:350-351.
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39
40. REFERENCES
31. Usmangani k Chhalotiya.; Kashyap k.Bhatt.; Dimal A. Shah.; Sunil L.Baldania.: Development & Validation of a Stability
Indicating RP-HPLC method for Duloxetine Hydrochloride in its Bulk and Tablet dosage forms: Scientia pharmaceutica,
2010,78,857-868.
32. Dantu Durga Rao.; Shakil S. Sait.; Mukkanti.: Analysis of Duloxetine Hydrochloride and Its Related Compounds in
Pharmaceutical Dosage Forms and In Vitro Dissolution Studies by Stability Indicating UPLC ,Journal of Chromatographic
Science, 2010,48 , 819-824.
33. Navneet kumar.; D.Sangeeta.; P.Balakrishna: Development & Validation of a UPLC method for the determination of
Duloxetine.Hydrochloride residues on pharmaceutical equipment surfaces , Pharmaceutical methods, 2011 ,2(3),161-166.
34. Veera Reddy.Arava.; Sreenivas ulareddy.; Bandat Makru.; kameshwar Rao cherukuri.; Madhusudhan reddy. : A Stability-Indicating
RP-HPLC Method for Development and Validation for Duloxetine Hydrochloride in Tablets ,Der pharma chemica , 2012
,4(4),1735-1741.
35. D.Chandrapal reddy.A.T.Bapuji.;V.Suryanarayana Rao Himabindu.D.; Development and Validation of a LC/MS/MS Method for
the Determination of Duloxetine in Human Plasma and its Application to Pharmacokinetic Study: E-Journal of Chemistry,2012,
9(2), 899-911.
36. Srinivasulu Dasari.; Raj kumar viriyala.; k santhosh.; Archana kumari.; A Validated RP-HPLC method for the analysis of
Duloxetine Hydrochloride in Pharmaceutical dosage forms: International Journal of Comprehensive Pharmacy,2010,3(03),1-3.
37. Narasimha rao R.; Laxmi Raj A.; Samjay kumar Ch.; kapil Ch.; chaitanya M; Method Development & Validation of Duloxetine
Hydrochloride by RP-HPLC; International Journal of Research in Pharmaceutical& Biomedical Sciences.2011,2(2),1335-1340.
38. http://en.wikipedia.org/wiki/Inductively_coupled_plasma_mass_spectrometry
39. ICH guideline, Q3D: Guideline for Metal Impurities.
40. Http;//Pubchem.Ncbion/M.Nih.Gov/Summary
40
41. ACKNOWLEDGEMENTS
Dr. M. V. SURYANARAYANA
Dr. S. GANANADHAMU
Dr. AHMED KAMAL
Dr. R. SRINIVAS
Dr. M. V. N. TALLURI
Dr. N. SATEESH KUMAR
Dr. P. K. JANA
Dr. DHARMENDRA
Mr. M. RAVIKUMAR 41
45. EXPERIMENTAL SECTION
Mobile phase A:
Buffer preparation: 0.77 gm of Ammonium Acetate buffer was accurately weighed transferred into 1litre milliQ water, sonicated to
dissolve and added 1ml of TEA, finally pH 6.6 adjusted with dilute Glacial acetic acid.
Mobile phase A: Buffer : ACN (90:10)(%v/v)
Accurately measured 900ml of buffer and 100 ml of Acetonitrile, and were transferred in to a mobile phase container mixed
thoroughly, finally sonicated to degas.
Mobile phase B: Acetonitrile: Methanol (60:40) (%v/v)
Accurately measured Acetonitrile, Methanol individually 600ml , 400ml respectively with 1litre measuring cylinder and were
transferred into mobile phase container, mixed thoroughly Sonicated to degass , finally 5ml water added to the above organic phase.
Column Washing Solution (ACN: Water 50:50%v/v)
500mL of milliQ water & 500ml of ACN were accurately measured individually with 500ml measuring cylinder, were taken and
filtered through 0.22μ filter, and mixed thoroughly to get solution of 50:50(% v/v.)
Strong needle wash solution (ACN: Water 90:10%v/v)
100 ml of Milli-Q water, 900ml of ACN were accurately measured individually with 1000ml measuring cylinder, filtered through
0.22μ filter, and mixed thoroughly to get final solution of 100:900% v/v of Water: Acetonitrile.
45
46. EXPERIMENTAL SECTION
Weak needle wash solution (ACN: Water 10:90%v/v)
900 ml of Milli-Q water, 100ml of ACN were accurately measured individually with 1000ml measuring cylinder, filtered through 0.22μ
filter, and mixed thoroughly to get final solution of 900:100% v/v of Water: Acetonitrile.
Standard stock solution (0.2mg/ml) : Accurately weighed and transfered about 20 mg of standard into a 100ml volumetric flask,
dissolved in and diluted to volume with diluent.
Spiked sample solution with impurities: Accurately weighed and transfered about 20 mg of standard was taken into 100ml
volumetric flask and make up the volume with Reference stock.
Reference stock solution: All individual impurities of 2.5mg each and standard of 2mg, individually weighed, transferred to 25 ml,
20ml volumetric flask respectively, finally diluted unto the mark with diluents to make their individual reference stock solutions.
Reference solution (100%): Pipetted out 0.3ml of each impurities from reference stock impurities,0.2ml of standard solution from
reference stock solution transferred into 100ml volumetric flask and diluted upto the mark with the diluent. Final impurities, standards
are of concentrations 0.0003252mg/ml (Di methyl impurity), 0.0003336mg/ml (Alcohol impurity), 0.0003156mg/ml (Naphthol
impurity), 0.0003372mg/ml (Impurity C) and 0.000215mg/ml (Duloxetine standard).
46
47. LITERATURE REVIEW
s.
no
.
Title of the article Column Mobile phase
composition
(%V/V)
Flow rate
(ml/mn)
Wave
Length
(nm)
1 Development and validation of Stability
indicating RP HPLC method for
Duloxetine HCl in its bulk and dosage
forms.
Hypersil
C-18 column (250mm
*4.6 mm id, 5μm )
Acetonitrile:
0.01Mpotassiumdihydrogen
phosphate buffer (pH 5.4 adjusted
with orthophosphoricacid)
(50:50, %v/v)
1.0
ml/min
229nm
2 Analysis of Duloxetine Hydrochloride
and Its Related Compounds in
Pharmaceutical Dosage Forms and
InVitro Dissolution Studies by Stability
Indicating UPLC
UPLC
C-18 column
50 mm 4.6 mm,
1.8 μm
Mobile phase A : 0.01M
potassium dihydrogen phosphate
(pH 4.0) buffer, tetrahydro furan,
and methanol
67:23:10 (v/v/v) Mobile phase B :
0.01 M potassium dihydrogen
phosphate, (pH 4.0) buffer,
Acetonitrile
60:40 (%v/v).
0.6
ml/min
236nm
3 Development and validation of uplc
method for determination of Duloxetine
HCl residues on pharmaceutical mfg
equipment surfaces .
UPLC HSS T3
100*2.1mm,1.7 μm
0.01M potassium dihydrogen
phosphate (pH3.0) buffer, and
Acetonitrile(60:40)( %v/v)
0.4
ml/min
230
4 Stability Indicating Nature of RP-HPLC
method for Determination of Impurity
profile and Degradation impurities in
Duloxetine Hydrochloride
YMC Pack C8, 250 *
4.6 mm, 5μm column
solvent A (0.01 M of Sodium Di
hydrogen Orthophosphate and
1.0g of 1-Heptane Sulfonic Acid
Sodium Salt transfer in 1000mL of
water, pH3.0 using Ortho
phophoric acid) and solvent B
(Acetonitrile).
1ml/min 217
5 Development and Validation of a
LC/MS/MS Method for the
Determination of Duloxetine in Human
Plasma and its Application to
Pharmacokinetic Study
X-terra RP8 (50
mm*4.6 mm, 5 μm
particle size) column
30 mM ammonium formate (pH-
5.0) and acetonitrile as an isocratic
0.4
ml/min
6 Avalidated RP- HPLC method for the
analysis of duloxetine hydrochloride in
pharmaceutical dosage forms
Inertsil BDS (250*4.6
mm) C8 column
Buffer: Acetonitrile: Methanol
(55:37:8%v/v)
1ml/min 215
nm
7 Method Development and Validation of
Duloxetine Hydrochloride by RP HPLC
BDS Hypersil C18
150 x 4.6 mm,
5mm
Buffer: Mixed phosphate
buffer(1.625g potassium di
hydrogen phosphate
+ 0.3g Di potassium hydrogen
phosphate
in 550ml water)
1ml/mim 232
47
48. CLASSIFICATI
ON
ORAL EXPOSURE PARENTERAL EXPOSURE INHALATION
EXPOSURE*
PDE(µg/day) CONCENTRATIO
N
(ppm)
PDE(µg/day) CONCENTRATIO
N
(ppm)
Class 1 A
Pt,Pd 100 10 10 1 Pt:70
Class 1 B
Ir,Rh,Ru,Os 100** 10** 10** 1**
Class 1 C
Mo,Ni,Cr,V
metals of
significant
toxicity
250 25 25 2.5 Ni:100
Cr(VI):10
Class 2
Mn,Cu
Metals with low
safety concern
2500 250 250 25
Class 3
Fe,Zn
Metals with
minimal safety
concern
13000 1300 1300 130
48
