Separation, Identification and Quantification of process related Impurities and Stress Degradants of Gefitinib by LC-ESI-Q–TOF/MS

 

Pramadvara Kallepalli*, Mukthinuthalapati Mathrusri Annapurna

Department of Pharmaceutical Analysis and Quality Assurance, GITAM Institute of Pharmacy, GITAM University, Visakhapatnam-530045, India

*Corresponding Author E-mail: pramadvarakallepalli4@gmail.com

 

ABSTRACT:

Gefitinib, an anticancer drug and its process related impurities were estimated using WATERS 2695 Series HPLC system equipped with Quaternary gradient pumps and WATERS 2996 PDA detector with EMPOWER software. ESI-QTOF- MS system with Waters Quattro premier XE triple quadrupole with Acquity HPLC system with Masslynx software was used for the mass spectral studyof the impurities. Reverse phase C-18 column-Inertsil ODS 3V (150 X 4.6 mm, 5µm particle) was used and the total run time was 45 min. Gefitinib was subjected to forced degradation studies and the degradant products were analyzed using mass spectroscopy. Impurities (I - X) were separated on isocratic mode and calibrated using mass spectroscopy. Gefitinib was found to be more sensitive towards oxidation and three degradants were observed and their mass spectra also analyzed. The method was validated (ICH guidelines).

 

KEYWORDS: Gefitinib;RP-HPLC; ESI-QTOF-MS; stability indicating; Impurities, validation, ICH guidelines.

 

 

 

INTRODUCTION:

Gefitinib (C22H24ClFN4O3) is an anticancer drug acts by binding on the cell surface and blocks cell division by interrupting the signals. Gefitinib is a tyrosine kinase inhibitor1-3. Gefitinib (Figure 1), 4-(3'-chloro-4'-Fluoroanilino)-7-methoxy-6-(3-morpholino propoxy) quinazoline is a white powder having molecular weight 446.902 g/mol and pKa values 5.4 and 7.2. Different instrumental techniques were used for the determination of Gefitinib 4-10 and its related substances as well as impurities 11-12 using HPLC and LC-MS in biological fluids13-16. A new stability indicating LC-MS method was established and 10 impurities were separated, calibrated and analyzed using their mass spectra in the proposed research work.

 

 

 

Figure 1: Chemical structure of Gefitinib

 

MATERIALS AND METHODS:

Gefitinib samples (>99.5 purity) and its impurities were procured from in-house TherdosePvt Ltd (Hyderabad, India).HPLC grade acetonitrile, ammonium acetate, hydrochloric acid, sodium hydroxide and 30% w/v hydrogen peroxide were obtained from Merck. Ultrapure water was obtained from Milli-Q Gradient Millipore system.Stock solutions of Gefitinib and its impurities (I-X) (1000 µg/ml) were prepared in acetonitrile and stored in refrigerator at 2-8ºC.

 

 

Chromatographic conditions:

Chromatographic study of Gefitinib and its impurities were analyzed by HPLC system WATERS 2695 Series equipped with a separation module, Quaternary gradient pumps, thermostatic column compartment, WATERS 2996 PDA detector and auto sampler withEMPOWER software for the separation of impurities and conducting stress degradation studies. ESI-QTOF-MS system Waters Quattro premier XE triple quadrupole with Acquity HPLC system withMasslynx software was used for the mass spectral study of the impurites.

 

10 mM of ammonium acetate: acetonitrile (63:37% v/v) (pH was adjusted to 6.5 with glacial acetic acid) was used as mobile phase with flow rate 1 mL/min and 10 µL of injection volume (detection wavelength 240 nm).Stress degradation studies were conducted on LC MS Agilent 6120 single quadrapole with Agilent 1200 infinity series HPLC. Reverse phase C-18 column- Inertsil ODS 3V (150 X 4.6 mm, 5µm particle) was usedthroughout the studies (column temperature 30°C).

 

Validation analytical method:

A series of Gefitinib17(0.2-750 µg/mL) solutions and also Gefitinib spiked along with all the10 impurities18 (I-X) (each 0.15% w/w) were prepared andinjected in to the system. The mean peak area was noted and calibration curves were builtusing mean peak area against concentration of analyte.

 

System precision (n=10), intraday (n=6) and inter day (n=6) precision studies were performed for Gefitinib spiked along with its ten impurities (I-X) (each 0.15% w/w) and the % RSD of mean peak area as well as the retention time (Rt) were determined. Accuracy study was performed for Gefitinib (0.02 µg/mL) as well as its impurities (0.0002µg/mL) different levels i.e. 50%, 100% and 150% to that of LOQ by the addition of API to a constant concentration of extracted tablet formulation solution and % RSD was determined.

 

Assay of Gefitinib tablets:

Gefitinib is available as tablets as well as film coated tablets (250 mg) with brand names Geftinat (NatcoPharma Limited), Gefonib (Miracalus Pharma Pvt Ltd), Geftib (GlenmarkPharmaceuticaks Ltd) (Onkos), Gefticare (Medicare Remedies Pvt Ltd) and Geftifos (Torrent Pharmaceuticals Ltd). 20 Tablets of three different brands were purchased from the Pharmacy and extracted with acetonitrile and later diluted with mobile phase as per necessity.

 

Stress degradation studies19

Gefitinib was exposed to three different stress conditions and their degradation pathway was studied with the help of mass spectral studies along with liquid chromatography.Acidic hydrolysis (5N HCl at 80°C for 1hour), alkaline hydrolysis (5N NaOH at 80°C for 1 hour) and oxidation (5% H2O2at 80°C for 1 hour) reactions were performedwith Gefitinib and those solutions were injected in to the system after neutralization. Mass spectroscopic studies were performed for Gefitinib and its impurities.

 

RESULTS AND DISCUSSION:

A sensitive stability indicating LC-ESI-Q-TOF/MS method has been establishedfor the determination of Gefitinib and its process related impurities were analyzed by LC-MS and calibrated(isocratic mode) in Gefitinib tablets. Mass spectra was used for the analysis of degradant products (DP) obtained during the stability studies. A review of previous literature on the analytical methods of Gefitinib was given in Table 1.

 

 

Table. 1.Comparison of present LC-MS method with the earlier reported methods

Mobile phase (v/v)

λ(nm)

Linearity (mg/ml)

Method

Ref

Methanol: Dihydrogen potassium phosphate (85:15)

247

0.14 - 0.52

HPLC

4

Methanol: Triflouroacetic acid(35:65)

246

-

HPLC

5

Acetonitrile: Phosphate buffer(55:45) (pH 3.6)

248

25-150

HPLC

6

Acetonitrile: phosphate buffer (pH 3.5) (Gradient mode)

210

0.0015-0.07

HPLC

7

Acetonitrile: Ammonium dihydrogen phosphate buffer(30:70)

205

50 - 150

HPLC

8

Methanol: Dipotassium Hydrogen ortho phosphate(10:90)

246

25-300

HPLC

9

Acetonitrile: Potassium di-hydrogen phosphate (55:45)pH 6.5

220

10-60

HPLC

10

Acetonitrile: 130mM Ammonium acetate and (37: 63); pH 5.0

(Impurities and Gefitinib)

260

0.1-2.0

25-500

HPLC

11

Acetonitrile:Ammonium acetate(40:60)

250

-

RRLC

12

Acetonitrile:  Ammonium acetate 0.5%(Gradient mode)

300

-

LC-MS/MS

13

Acetonitrile: water (70:30) 0.1% formic acid(Isocratic mode)

(Human plasma, Mouse plasma and Tissues)

-

-

LC/MS/MS

14

Acetonitrile: water (50:50)(Human serum, CSF)

-

LC/MS/MS

15

Acetonitrile: water(0.1% formic acid)(Mouse plasma)(Gradient mode)

-

-

LC-MS/MS

16

10 mMAmmonium acetate: Acetonitrile (37:63%)(Isocratic mode)

(pH adjusted to 6.5 with acetic acid)(10 Impurities and Gefitinib)

240

0.2 - 750

LC-ESI-Q-TOF/MS

and 10 Impurities

Present method

 

Method optimization:

Initially many trails were made to analyze Gefitinib API and later to separate the process related impurities (I-X) using ammonium acetate buffer and acetonitrile as mobile phase. pH of the aqueous phase in combination with acetonitrile place a very important role in separation and elution of Gefitinib, its impurities as well as the degradants products obtained during the stability studies. 10 mM of ammonium acetate buffer and acetonitrile mixture maintained at pH 6.5 with the help of acetic acid was finally selected (63:37%v/v)for the entire study within 45 min run time with flow rate 1.0 ml/min (UV detection at 240 nm). Gefitinib was eluted at 28. 260 min along with other 10 impurities (Figure 2).

 

 

 

 

(a)

(b)

 

(c)

Figure 2: Representative chromatograms of a) Blank b) Gefitinib (1000 µg/mL) c) Gefitinib spiked with 0.2% of impurities

 

 

 

METHOD VALIDATION:

Gefitinib and the impurities (I-X) follows linearity over the concentration range 0.2-750µg/mLand 0.2–5 µg/mL for impurities (Table 2). The % RSD in system precision (Table 3), intraday (Table 4), inter day (Table 5) precision and accuracy (Table 6) studies was less than 2.

 

 

 

 

 

 

 

Table. 2.Linearity ofGefitinib and its impurities

Sample name

(Impurities / Drug)

Regression equation

(Correlation coefficient, r2)

I

Y=35531x+76.51 (0.9997)

II

Y=69079x+1263.9 (0.9995)

III

Y=35978x+244.65 (0.9998)

IV

Y=31193x+167.01 (0.9999)

V

Y=28696x+26.739 (0.9997)

VI

Y=33571x+150.19 (0.9998)

VII

Y=37925x+405.62 (0.9997)

VIII

Y=27219x+682.51 (0.9993)

IX

Y=27916x+169.19 (0.9998)

X

Y=85033x+190.27 (0.9999)

Gefitinib

Y = 4084.2x + 13939 (0.9999)

Limits of detection (LOD); Limit of quantification (LOQ)

 

Table. 3. System precision study of Gefitinib and its impurities

Sample name (Impurities/ Drug)

*Rt (min)

% R.S.D.

*Mean peak area

% R.S.D.

I

5.61

0.89

84803

0.54

II

14.05

0.36

160247

0.35

III

15.50

0.19

91047

0.86

IV

16.59

0.24

67953

0.34

V

21.29

0.23

59893

0.75

VI

23.52

0.17

72324

0.41

VII

25.32

0.20

78114

0.32

VIII

27.37

0.10

55668

0.46

IX

30.44

0.30

58469

0.63

X

33.70

0.10

163559`

0.25

Gefitinib

28.20

0.28

174191

0.51

*Mean of ten determinations

Table. 4. Intraday precision study of impurities

Impurities

1

2

3

4

5

6

*Mean peak area±SD (%RSD)

I

84803

84252

83983

84591

84950

85078

84609.5±422.7353 (0.50)

II

170247

169459

172542

175011

168287

169356

170817±2500.79(1.46)

III

91047

92000

91456

91250

91802

90546

91350.17±526.5482(0.58)

IV

72596

71970

72552

73456

72496

71956

72504.33±548.1962(0.76)

V

60292

59521

58615

59512

61050

60920

59985±940.0664(1.57)

VI

72579

71920

72153

72541

71456

72345

72165.67±426.1501(0.59)

VII

80398

81236

79850

78560

79956

81023

80170.5±965.0123(1.20)

VIII

57966

58023

57654

58231

57356

58021

57875.17±315.0996(0.54)

IX

59269

58569

58456

59241

58461

60253

59041.5±701.76(1.19)

X

171559

170523

175231

174253

175241

173250

173342.8±1956.464(1.13)

Table. 5. Intermediate precisionstudy of impurities

Impurities

Day 1

Day 2

Day 3

Day 4

Day 5

Day 6

*Mean peak area± SD(%RSD)

I

85803

83150

80520

83591

83950

84078

83515.33±1723.378(2.06)

II

172247

168329

171259

174915

169320

168741

170801.8±2520.85(1.48)

III

91251

92456

92300

93250

92510

92351

92353±641.4178(0.69)

IV

72456

72510

72456

75820

73102

72560

73150.67±1330.577(1.82)

V

62652

62531

59561

65420

66020

62360

63090.67±2345.415(3.72)

VI

71579

72100

73210

72651

72056

73254

72475±677.797(0.94)

VII

82395

82360

82561

84230

82953

83023

82920.33±699.4145(0.84)

VIII

58980

58535

58562

59420

58356

58420

58712.17±409.6137(0.70)

IX

61296

59512

62590

60592

60236

62360

61097.67±1214.281(1.99)

X

172590

169230

176520

175230

175230

171231

173338.5±2798.538(1.61)

Table. 6. Accuracy study of Gefitinib and its impurities

Sample Name

(Impurities/ Drug)

% Recovery

LOQ

50%

100%

150%

I

95.8 ± 1.3

91.7±1.4

104.2±0.5

97.2±0.5

II

104.3±0.2

98.2±1.8

95.7±1.2

102.8±0.9

III

98.0±2.5

96.8±2.8

99.2±1.8

98.7±3.0

IV

106.5±3.3

102.6±0.8

103.5±0.8

102.3±1.3

V

105±2.9

99.0±2.3

104.8±0.3

99.4±2.1

VI

97.4±0.6

96.3±0.9

101.2±2.9

96.0±0.8

VII

95.2±3.5

99.0±2.3

102.4±0.7

98.7±1.6

VIII

104.8±2.8

99.1±2.7

100.9±2.2

96.9±2.4

IX

104.8±0.2

99.0±1.5

100.5±0.6

99.0±0.25

X

102.4±2.8

103±0.4

105.5±0.8

103.7±2.8

Gefitinib

97.4±0.96

98.2±1.21

100.2±0.74

99.2±0.42

Table. 7. System suitability of Gefitinib and its impurities

Sample name (Impurities/ Drug)

*Rt± SD

%R.S.D.

Relative retention time

Tailing factor

Theoretical plates

I

5.61±0.0500

0.89

0.199

1.158

85721

II

14.05±0.0660

0.36

0.498

1.216

69821

III

15.50±0.0297

0.19

0.550

1.133

59249

IV

16.59±0.0411

0.24

0.588

1.219

48724

V

21.29±0.0510

0.23

0.754

1.346

39854

VI

23.52±0.0396

0.17

0.834

1.215

56723

VII

25.32±0.0461

0.20

0.897

1.321

78237

VIII

27.37±0.0195

0.10

0.971

1.168

93481

IX

30.44±0.0910

0.30

1.079

1.317

16234

X

33.70±0.0233

0.10

1.195

1.102

54892

Gefitinib

28.20±0.0814

0.28

1.000

1.204

49628

 

System suitability and solution stability of Gefitinib and its impurities (25°C):

System suitability was determined by injecting Gefitinib 1000µg/mL with 0.15% level of all impurities and the results were shown in Table 7. Stability of Gefitinib standard solution was determined by injecting the standard Gefitinib solutions at different time intervals (1, 15, 20, 24 and 48 hrs) and found that the solutions are very much stable (Table 8).  Stability of Gefitinib impurities solutions was determined by injecting the impurities solutions at different time intervals (1, 15, 20, 24 and 48 hrs) and found that the solutions are very much stable (Table 9).

 

Table. 8.Solution stability of Gefitinib (25°C)

Time (hrs)

Peak area

% Area variation

Initial

178230

NA

1

180230

-2.56

15

180365

-2.73

20

180265

-2.60

24

180560

-2.98

48

178510

-0.36

Table. 9. Solution stability of Gefitinib impurities (25°C)

Impurities

Time(hrs)

Peak area

% Area variation

I

0

85562

NA

1

84692

1.02

15

86952

-1.62

20

85651

-0.10

24

86541

-1.14

48

89561

-4.67

II

0

169427

NA

1

168591

0.49

15

169592

-0.10

20

168562

0.51

24

169752

-0.19

48

168852

0.34

III

0

92589

NA

1

91956

0.68

15

91985

0.65

20

92568

0.02

24

94859

-2.45

48

96232

-3.93

IV

0

74586

NA

1

73568

1.36

15

74589

0.00

20

77456

-3.85

24

75683

-1.47

48

75961

-1.84

V

0

63256

NA

1

62596

1.04

15

63568

-0.49

20

63598

-0.54

24

64589

-2.11

48

64589

-2.11

VI

0

71256

NA

1

70956

0.42

15

73906

-3.72

20

74581

-4.67

24

71698

-0.62

48

72691

-2.01

VII

0

80398

NA

1

81956

-1.94

15

80239

0.20

20

81420

-1.27

24

82390

-2.48

48

84230

-4.77

VIII

0

58590

NA

1

58423

0.29

15

59410

-1.40

20

57985

1.03

24

59415

-1.41

48

56869

2.94

IX

0

61236

NA

1

62136

-1.47

15

62587

-2.21

20

62189

-1.56

24

61258

-0.04

48

60985

0.41

X

0

172596

NA

1

175892

-1.91

15

174568

-1.14

20

167895

2.72

24

169741

1.65

48

170589

1.16

 

Stress degradation studies:

Gefitinib (Rt 28.260 min) was subjected to forced degradation i.e. acidic (Rt 28.063 min) and alkaline hydrolysis (Rt 28.208 min) and the solutions were neutralized, diluted and chromatographic study was continued and no degradants were found indicating that Gefitinib is highly resistant towards acidic (1.67) and alkaline (1.17) environment while durig oxidation a significant degradation of the drug was observed (11.98) (Table 10). Gefitinib undergoes degradation and three degradants were eluted at (Rt 8.031, 13.412, 16.665 min) and therefore more emphasis was given for the degradants eluted and mass spectroscopy was performed for oxidation study. The chromatograms and mass spectra obtained were shown in Figure 3 and Figure 4. The plausible oxidation degradation pathway of Gefitinib was shown in Figure 5.

 

 

Table.10. Stress degradation studies of Gefitinib

Stress condition

Rt (min)

m/z

% Degradation

Tailing Factor

Theoretical  plates

Gefitinib

28.260

448.2

-

1.204

49628

Acidic degradation (5N HCl /80°C/ 1 hr)

28.063

-

1.67

1.128

52465

Alkaline degradation (5N NaOH / 80°C/ 1hr)

28.208

-

1.17

1.362

46892

Oxidative degradation (5%H2O2 /80°C /1hr)

28.301

8.031 (DPI)

13.412 (DPI)

16.665 (DPI)

448.2

222.0

287.1

248.1

11.98

1.119

51264

 

Specificity:

The specificity was checked by stress degradation studies. Gefitinib drug peak was well separated in acidic, alkaline and oxidative degradation condition. During oxidation Gefitinib produced three new degradation products (DP1, DP2 and DP3) and which were well eluted without interfering with the drug peak with resolution greater than 2 and the above information proves that the method is selective and specific. The mass spectra of DPs were not at all match able with the 10 impurities already worked out and hence it is concluded that there are three unknown DPs still for the future analysts to project. The mass spectra of the 10 impurities was shown in Figure 6.

 

 

 

 

GefitiniB

Acid degradation

 

 

Alkaline degradation

Oxidative degradation

Figure 3: Chromatograms of Gefitinib during stress degradation studies

 

 

 

Gefitinib(C22H24ClFN4O3) (calculated m/z=447.59)

DP-I (C10H5ClFN3) (calculated m/z=223.10) at RT 8.031

 

 

DP-II (C15H19N3O2) (calculated m/z=287.10)at RT 13.412

DP-III (C14H19N03) (calculated m/z = 248.10) at RT 16.665 min

 

Figure 4 : Mass spectra of Gefitinib and its degradation products during oxidation

 

 

Figure 5: Plausible degradation pathway of Gefitinib during oxidative degradation

 

 

Mass spectrum of Impurity I (C29H37ClFN504) (m/z = 575.54)

 

Mass spectrum of Impurity II (C15H11ClFN3O2) (m/z = 320.45)

 

Mass spectrum of Impurity III (C6H5Cl2N) (m/z = 160.86)

 

Mass spectrum of Impurity IV (C22H24ClFN4O4) (m/z = 463.53)

 

Mass spectrum of Impurity V (C22H25FN4O3) (m/z = 413.61)

 

Mass spectrum of Impurity VI (C16H21N3O4) (m/z = 320.58)

 

Mass spectrum of Impurity VII (C21H22ClFN4O3) (m/z = 431.64)

 

Mass spectrum of Impurity VIII (C22H25ClN4O3) (m/z = 429.56)

 

Mass spectrum of Impurity IX (C22H24Cl2N4O3) (m/z = 463.53)

 

Mass spectrum of Impurity X (C21H22ClFN4O3), m/z = 432.46

 

Mass spectrum of Gefitinib (C22H24ClFN4O3) m/z = 447.59

Figure 6: ESI Mass spectra for Gefitinib and its process related impurities

 

 

 

CONCLUSIONS:

A simple validated stability indicating LC-ESI-Q-TOF/MS was developed for the determination ofGefitiniband its process c.Gefitinib was observed to be quite resistant towards alkaline and acidic hydrolysis while it remained unstable towards oxidative stress conditions. Three DPs were separated selectively by the proposed LC-ESI-Q-TOF/MS experiment. The fragmentation pathway of Gefitinib was also outlined and can be useful for the characterization of drug-drug interactionstudy and drug-excipient interaction studies as well as for the metabolite study. The proposed LC-ESI-Q-TOF/MS method was validated as per ICH guidelines and found to be sensitive, selective and specific.

 

ACKNOWLEDGEMENT:

The authors are grateful to Suven Life Sciences (India) for supporting this analysis and Therdose Pvt Ltd (Hyderabad, India) for providing the gift samples of Gefitinib and its process related impurities. There is no conflict of interest.

 

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Received on 07.08.2018          Modified on 22.08.2018

Accepted on 25.08.2018        © RJPT All right reserved

Research J. Pharm. and Tech 2018; 11(8): 3647-3657.

DOI: 10.5958/0974-360X.2018.00672.8