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Development And Validation Of LC-MS Compatible UPLC RS Method For Duloxetine.HCl Using QbD
&
Impurity Profiling Of Milnacipran.HCl By ICP-MS
Presenting ByGaneshwari Eedunuri
NIPER,HYD.
GuideDr. M.V.SuryanarayanaVice-PresidentMYLAN Laboratories,Ltd
Co GuideDr. S.GananadhamuAssistant professorNIPER,Hyderabad
1
CONTENTS
INTRODUCTION
AIM AND OBJECTIVE
DRUG PROFILE
METHOD DEVELOPMENT
METHOD VALIDATION
CONCLUSION
2
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
• More samples analysed per system or per scientist.
• Traceability
• Environmental friend
Chapter-1
DEVELOPMENT AND VALIDATION OF DULOXETINE.HCl
RS METHOD BY UPLC USING QbD
3
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
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
S. no.
Title of the article Column Mobile phase composition(%V/V)
Flow rate
(ml/min)
Wave Length(nm)
References
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)
1.0ml/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)
0.4ml/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
0.4ml/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 232nm37
LITERATURE REVIEW
6
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
EXPERIMENTAL
MATERIALS , REAGENTS: SUPPLIERS:
Duloxetine.HCl and its impurities Mylan pharma ltd,
Ammonium acetate buffer Rankem
Acetonitrile Rankem
Methanol Lichrosolv Merck
Triethylamine Rankem
Milli-Q water Milli-Q gradient A10
Filter membrane Whattman paper(G.V 0.22µ)
Hydrogen peroxide Rankem
Hydrochloric Acid Rankem
Sodium Hydroxide Rankem
8
Initial Screening conditions
16.a.1.a trial chromatogram
9
Figure : Design space region showing the dependent effects of ACN and % strong solvent on method success.
Design Space
10
Mobile phaseA: 10mM Ammonium Acetate buffer with 0.1% TEA ,pH 6.6 adjusted with dilute Glacial acetic acid.,finally added 10%ACNMobile Phase-B: ACN (100%v/v). Chromatographic parameters:Column : Cosmicsil Abra C8(50x2.1 mm,1.8 µ.) Detector : 230nmFlow rate : 0.8ml/min.Injector volume : 0.4 µl.Column oven temp : 50ºc.Sample cooler temp : 10ºc.Run time : 13 min.Diluent : Water : ACN (75:25%v/v).
Time(min) Flow rate(ml/min) %A %B
0 0.8 70 30
0.2 0.8 70 30
3 0.8 60 40
5 0.8 50 50
8 0.8 20 80
9 0.8 70 30
13 0.8 70 30
Method optimisation Trials
Mobile Phase-B: ACN : Methanol(90:10%v/v))
Inference:One of the impurity coeluting with the standard peak.Inference:Resolution is less between Napthol impurity and Duloxetine standard.
Trial-1Trial-2
Gradient run:
11
Mobile phase A: 10mM Ammonium Acetate buffer with 0.1% TEA, pH 6.6 adjusted with dilute Glacial acetic acid,finally added 10%ACN.Mobile phase B: Acetonitrile : Methanol (80:20(%v/v))Chromatographic parameters:Column: Cosmicsil AbraC8(50x2.1 mm,1.8 µ.). Detector: 230nm.Flow rate: 0.8 ml/min.Injector volume: 0.4µl.Column oven temp: 50 c.Sample cooler temp: 10c.Run time: 13 min.Diluent: Water : ACN (75:25%v/v).
Gradient run:
Trial-3
Time(min) Flow Rate(ml/min)
%A %B
0 0.8 60 40
0.2 0.8 60 40
3 0.8 55 45
5 0.8 50 50
8 0.8 20 80
9 0.8 60 40
13 0.8 60 40
Inference: Resolution is less between Naphthol impurity and Duloxetine.HCl standard
Column oven temp: 40 c.
Trial-4
Inference: Resolution between Naphthol impurity and Duloxetine.HCl standard is 2.2
12
Mobile phase A: 10mM Ammonium Acetate buffer with 0.1% TEA ,finally pH 6.6 adjusted with diluted Glacial acetic acid ,finally added 10%ACN.
Mobile phase B: Acetonitrile : Methanol (60:40(%v/v))Chromatographic parameters:Column: Cosmicsil Abra C8(50x2.1 mm,1.8 µ.). Detector: 230nm Flow rate: 1 ml/min.Injector volume: 4 µl.Column oven temp: 40c.Sample cooler temp: 20c.Run time: 8 min.Diluent: Water : ACN (75:25%v/v).
Time(min) Flow rate(ml/min) %A %B
0 1 60 40
0.5 1 60 40
2.4 1 55 45
5.5 1 50 50
6 1 60 40
8 1 60 40
Trial-5
Inference: Resolution between Naphthol impurity and Duloxetine standard is 2.21
Gradient run:
Time(min) Flow rate(ml/min) %A %B
0 0.75 60 40
1 0.75 60 40
2 0.75 55 45
5.5 0.75 50 50
6.0 0.75 60 40
8 0.75 60 40
Trial-6
Inference: Di methyl impurity eluted at the re equilibration time.
Flow rate: 0.75 ml/min
13
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
Degradation Conditions
% Increase Of Alcoholimpurity
RRT-0.24
% Increase Of
Impurity CRRT-0.52
% Increase Of Naphthol Impurity
RRT-0.81
% Increase OfDi Methyl
ImpurityRRT-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
Degradation summary
Degradation Conditions
% Increase Of Alcoholimpurit
yRRT-0.24
% Increase Of
Impurity CRRT-0.52
% Increase Of Naphthol
ImpurityRRT-0.81
% Increase OfDi Methyl
ImpurityRRT-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 5days Chromatogram of Control Sample (solid) exposed to UV light for 5 daysChromatogram of Control Sample (solid) exposed to 105°C for 5days
16
ALCO
HOL I
MP - 0
.811
PurityAuto Threshold
AU
Degre
es
0.000
0.010
0.020
0.030
0.040
0.050
0.060
0.070
0.080
0.00
10.00
20.00
30.00
40.00
50.00
60.00
70.00
80.00
90.00
Minutes
0.70 0.72 0.74 0.76 0.78 0.80 0.82 0.84 0.86 0.88 0.90 0.92 0.94 0.96 0.98
NAPT
HOL IM
P - 2.6
98PurityAuto Threshold
AU
Degre
es
0.00
0.10
0.20
0.30
0.40
0.50
0.60
0.00
2.00
4.00
6.00
8.00
10.00
Minutes
2.52 2.54 2.56 2.58 2.60 2.62 2.64 2.66 2.68 2.70 2.72 2.74 2.76 2.78 2.80 2.82 2.84 2.86 2.88
IMP C
- 1.55
4
PurityAuto Threshold
AU
Degre
es
0.00
0.05
0.10
0.15
0.20
0.25
0.30
0.00
2.00
4.00
6.00
8.00
10.00
12.00
14.00
16.00
18.00
20.00
Minutes
1.42 1.44 1.46 1.48 1.50 1.52 1.54 1.56 1.58 1.60 1.62 1.64 1.66 1.68 1.70 1.72 1.74
DXT S
TD - 3
.030
PurityAuto Threshold
AU
Degre
es
0.00
0.05
0.10
0.15
0.20
0.25
0.00
10.00
20.00
30.00
40.00
50.00
60.00
70.00
80.00
90.00
Minutes
2.84 2.86 2.88 2.90 2.92 2.94 2.96 2.98 3.00 3.02 3.04 3.06 3.08 3.10 3.12 3.14 3.16 3.18 3.20 3.22
DI ME
THYL
IMP -
4.647
PurityAuto Threshold
AU
Degre
es
-0.02
0.00
0.02
0.04
0.06
0.08
0.10
0.12
0.14
0.16
0.18
0.20
0.00
10.00
20.00
30.00
40.00
50.00
60.00
70.00
80.00
90.00
Minutes
4.40 4.45 4.50 4.55 4.60 4.65 4.70 4.75 4.80 4.85 4.90
Specificity-Impurity interference
17
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 C1.8 1.8
3.
Naphthol impurity 2.8 2.8
4.
Duloxetine.HCl3.5 3.4
5. Dimethyl impurity5.3 5.3 Chromtogram of reference solution
Chromtogram of 100% spike solution
Chromtogram of blank
18
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
LOD ResultsLOQ Results
S.NO. Name Retention Time
Area%
AreaHeight
RT Ratio
USP Resolution
USP Tailing
USP Plate Count
1ALCOHOL
IMP0.83 261 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
3NAPHTHOL
IMP2.69 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
5DI METHYL
IMP5.16 120 2.42 15 1.529 8.8 0.9 7529.00
S.no.
NameRetention
TimeArea % Area Height
RT Ratio
USP Resolution
USP Tailing
USP Plate
Count
1ALCOHOL
IMP0.81 643 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
3NAPHTHOL
IMP2.69 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
5DI METHYL
IMP5.20 535 6.21 56 1.534 6.2 0.9 1999.08
20
Linearity of Alcohol impurity
y = 11049015.3893x - 294.5794
R2 = 0.9984
0.0
1000.0
2000.0
3000.0
4000.0
5000.0
6000.0
0.000000 0.000100 0.000200 0.000300 0.000400 0.000500 0.000600Conc(mg/mL)
Peak
area
Level
Conc.(mg/ml) Peak area
LOQ 0.000083 653.0
30% 0.000100 866.5
50% 0.000167 1572.5
75% 0.000250 2342.0
100% 0.000334 3337.0
125% 0.000418 4318.5
150% 0.000500 5311.0
Slope 11049015.39
Intercept -294.57936
R2 0.9992
Linearity
Level Conc.(mg/ml) Peak area
LOQ 0.000081 422.0
30% 0.000150 1515.5
50% 0.000163 2316.0
75% 0.000244 4593.0
100% 0.000325 6545.5
125% 0.000406 8573.0
150% 0.000488 10832.5
Slope 26101909.35
Intercept -1953.57868
R2
0.9983
Level Conc.(mg/ml) Peak area
LOQ 0.000069 4055.5
30% 0.000095 4996.0
50% 0.000158 8885.0
75% 0.000237 12988.5
100% 0.000316 17612.0
125% 0.000395 21820.0
150% 0.000473 27360.0
Slope 57160561.56
Intercept -266.14084
R2 0.999
Acceptance criteria:The plot of concentration versus peak area for each impurity and Duloxetine should be linear with R2 not less than 0.990.
Conclusion: The R2was found to be within the limits and the results were satisfactory.
Linearity of Duloxetine Hcl
y = 22163348.1200x - 114.4636
R2 = 0.9984
0.0
2000.0
4000.0
6000.0
8000.0
0.000000 0.000100 0.000200 0.000300 0.000400
Conc(mg/mL)
Peak
area
Level Conc.(mg/ml) Peak area
LOQ 0.000047 860.5
30% 0.000065 1456.5
50% 0.000108 2134.0
75% 0.000161 3142.5
100% 0.000215 5291.5
125% 0.000269 5879.0
150% 0.000323 7753.5
Slope 22163348.12
Intercept -114.46360
R2
0.9992
Level Conc.(mg/ml) Peak area
LOQ 0.000084 1044.0
30% 0.000101 1522.0
50% 0.000169 3406.0
75% 0.000253 5342.0
100% 0.000337 7659.5
125% 0.000421 9332.5
150% 0.000506 11788.0
Slope 25084314.66
Intercept -977.71720
R2 0.9993
Linearity of Napthol impurity
y = 57160561.5635x - 266.1408
R2 = 0.9983
0.0
5000.0
10000.0
15000.0
20000.0
25000.0
30000.0
0.000000 0.000100 0.000200 0.000300 0.000400 0.000500Conc(mg/mL)
Peak
area
Linearity of Di methyl impurity
y = 26101909.3527x - 1953.5787
R2 = 0.9966
0.0
2000.0
4000.0
6000.0
8000.0
10000.0
12000.0
0.000000 0.000100 0.000200 0.000300 0.000400 0.000500 0.000600Conc(mg/mL)
Peak
area
Linearity of impurity C
y = 25084314.6563x - 977.7172
R2 = 0.9986
0.0
2000.0
4000.0
6000.0
8000.0
10000.0
12000.0
14000.0
0.000000
0.000100
0.000200
0.000300
0.000400
0.000500
0.000600
Conc(mg/mL)
Peak
area
21
Accuracy Results
Set No. Level Conc. added(µg /ml) % Recovery
1
LOQ
Alcohol impurity
Imp C
Naphthol impurity
Dimethyl impurity
Alcohol impurity Imp C Naphthol
impurity Dimethyl impurity
0.083 0.084 0.069 0.081
117.5 96.1 106.6 104.6
2 113.9 92.5 110.9 109.5
3 113.9 92.5 110.9 97.7
1
50% 0.166 0.168 0.157 0.162
97.1 99.1 97.6 98.4
2 97.7 103.2 103.9 98.4
3 102.5 99.1 103.1 97.2
1
100% 0.333 0.337 0.315 0.325
100.1 97.9 94.7 92.7
2 103.1 97.0 95.1 94.7
3 103.4 98.5 95.7 91.9
1
150% 0.500 0.505 0.473 0.487
97.3 97.9 100.3 105.6
2 98.7 104.5 100.8 98.9
3 102.1 104.1 96.3 101.4
22
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 C97 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 110.90.53 to
3.422.37
Dimethyl impurity 92.7 to 105.6
97.7 to 109.51.72 to
1.522.62
23
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
Alcoholimpurity
Impurity C
Naphthholimpurity
Di methylimpurity
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
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
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
183.030.00 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
Parameters
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
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
Plasma generatespositive ions
Detector (e.g. electron multiplier)
Sorted by mass analyser, e.g. quadrupole, magnetic sector,
according to m/z ratioSpray chamber
sample
Nebuliser Interface
Under vacuum
Chapter-2
INTRODUCTION
IMPURITY PROFILING OF MILNACIPRAN.HCL BY ICP-MS
29
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 APt,Pd 100 10 10 1 Pt:70
Class 1 BIr,Rh,Ru,Os 100** 10** 10** 1**
Class 1 CMo,Ni,Cr,V metals of
significant toxicity
250 25 25 2.5 Ni:100Cr(VI):10
Class 2 Mn,Cu
Metals with low safety concern
2500 250 250 25
Class 3Fe,Zn
Metals with minimal safety concern
13000 1300 1300 130
30
System suitability
S.No
Element Standard conc.
(in ppb)
Check standard conc.
(in ppb)
%Recovery Correlationcoefficient
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.No.
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
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
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
Correlationcoefficient
--0.9997
Slope -- 1717.8806
Intercept -- -1445.4129
Linearity of Aluminium
y = 1717.9x - 1445.4
R2 = 0.9994
0
50000
100000
150000
200000
250000
300000
350000
400000
0 50 100 150 200 250
ConC (ppb)
Acceptance criteria: The correlation coefficient should not be less than 0.99.
Inference: Results obtained were within the limits.
33
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
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
Prep. No.
Aluminium (% Recovery)
Analyst-1/Day-1 Analyst-2/Day-2
1 103.091.0
2 103.293.3
3 119.090.2
4 106.591.7
5 105.393.0
6 109.593.0
Mean 107.892.0
SD 6.00761.266
%RSD 5.61.4
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
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
REFERENCES
1. Satinder Ahuja Henrik Rasmussen: Hplc Method Development For Pharmaceuticals ,2007,1st Edition:452-455.
1. ICH guideline, Q3A (R2) Impurities in New Drug Substances, Food and Drug Administration, USA, February 2006.
2. ICH guideline, Q3B (R2) Impurities in New Drug Products, Food and Drug Administration, USA, February 2006.
3. ICH guideline, Q3C (R4) Impurities in New Drug Products, Food and Drug Administration, USA, February 2005.
4. Renusolanki: Impurity profiling of active pharmaceutical ingredients and finished products, International Journal of Drug
Research Technologies ,2012, 3,231-238.
5. V.S.Tegeli,g.b.Gageli: Singnificance of impurity profiling , International Journal of Drug formulation & Research ,2011, 2,174-
195.
6. Gorog, S; Identification and determination of impurities in drugs, 1st edition ; Elsevier Science: 2000: 1-10.
7. Smith, R. J.; Webb, M. L: Analysis of drug impurities,1st edition; Wiley-Blackwell: 2008:1- 2
8. Kavitapilaniya.; harish.k: Recent trends in impurity profile of pharmaceuticals, Journal of Advanced pharm Technological
Research, 2010,1(3),302-310.
9. Duane A. Pierson.; Bernard A. Olsen.; David K. Robbins.; Keith M. DeVries.; and David L. Varie: Approaches to Assessment,
Testing Decisions, and Analytical Determination of Genotoxic Impurities in Drug Substances; Organic Process Research &
Development ,2009, 13, 285–291.
10. S.J.Ingale.; Chandra mohan sahu: Advance approaches for the impurity profiling of pharmaceutical drugs, International Journal
of Pharma & Life Sciences, 2011, 2 , 955-962.
11. Lioyd R.Snyder: Practical Hplc Method Development,3rd Edition :751-780.
12. 13. Jens.T.Carstensen.; C.T Rhodes : Drug Stability Principles & Practices 3rd Edition, Volume 107:306-375.
13. 14. Michael E. Swartz: UPLC: An Introduction and Review; Journal of Liquid Chromatography & Related Technologies, 2005,
28,1253-1263.
14. 15. Swartz.; M. E.; Murphy, B. J: Ultra performance liquid chromatography: tomorrow’s HPLC technology today. Lab Plus
International ,2004,18 (3): 6-9.
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REFERENCES
16. B. Srivastava.; B. K. Sharma.; Uttam Singh Baghel, Yashwant, Neha Sethi: Ultra performance liquid chromatography (UPLC):
A Chromatographic technique ; International Journal of Pharmaceutical Quality Assurance 2010,2(1), 19-25.
17. http://www.waters.com.
18. ICH guideline, Validation of analytical procedures, text and methodology Q2 (R1)
19. www.chromatographyonline.com
20. www.chromatography.com
21. Zarrin Es haghi: Photodiode Array Detection in Clinical Applications; Quantitative Analyte Assay Advantages, Limitations
and Disadvantages.
22. V.Kalyana Chakravarthy.; G. Kishore Babu.; R. Lakshmana Dasu.; P. Prathyusha and G. Aparna Kiran: The role of relative
response factor in related substances method development by HPLC; Royal Journal of Chemistry;2011,4,919-943.
23. http://www.sigmaaldrich.com/etc/medialib/docs/Aldrich/General_Information/lc_ms_brochure.
24. Devesh A. Bhatt.; Smitha I.Rane : QbD Approach to analytical RP-HPLC method development and its
validation :International Journal of Pharmaceutical Sciences, 2011,3,179-187.
25. Adrian A. Ammann: Inductively coupled plasma mass spectrometry (ICP MS): a versatile tool; Journal of Mass Spectrometry,
2007,42, 419–427.
26. ICH guideline, Q1A (R2) Stability Testing Of New Drug Substances and Products, USA, February 2003.
27. Http;//Pubchem.Ncbion/M.Nih.Gov/Summary
28. Merck Index: An Encyclopedia of Che mical Drugs & Biologicals,12th edition, 3514-3516.
29. Sean C Sweet Man.; Fr Phams. Matrindale.; The Complete Drug Reference,2007,5th Edition:350-351.
30. Deepak P.; Kumar Tn: Duloxetine Pharmacological Aspects: International Journal Of Biological &Medical Research
2011,2(2):589-592.
39
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
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
THANK YOU
42
43
44
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
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
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
CLASSIFICATION
ORAL EXPOSURE
PARENTERAL EXPOSURE INHALATION
EXPOSURE*
PDE(µg/day) CONCENTRATION
(ppm)
PDE(µg/day) CONCENTRATION
(ppm)
Class 1 APt,Pd 100 10 10 1 Pt:70
Class 1 BIr,Rh,Ru,Os 100** 10** 10** 1**
Class 1 CMo,Ni,Cr,V
metals of significant
toxicity
250 25 25 2.5 Ni:100Cr(VI):10
Class 2 Mn,Cu
Metals with low safety concern
2500 250 250 25
Class 3Fe,Zn
Metals with minimal safety
concern
13000 1300 1300 130
48