Project ppt

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).


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:


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


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


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


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


R2

0.9983


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


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


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


R2

0.9992


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


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:


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:


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:


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.

38

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

http://www.waters.com/

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

Documents

Project ppt