INTRODUCTION:

Gefitinib is an anti-cancer drug with molecular formula C₂₂H₂₄ClFN₄O₃ and molecular weight 446.902 g/mol. Gefitinib is chemically 4-(3'-chloro-4'-fluoroanilino)-7-methoxy-6-(3-morpholino propoxy) quinazoline (Figure 1) with pKa values 5.4 and 7.2 and it binds on the cell surface and blocks the cell division by interrupting signals and it is a tyrosine kinase inhibitor^1-3. Several instrumental techniques and analytical methods such as LC-MS/MS, RRLC and HPLC were used for the estimation of Gefitinib and its impurities in pharmaceutical formulations and in biological fluids.

Zheng⁴ et al., developed a LC-MS-MS method for the quantitative determination of Gefitinib in human serum and cerebrospinal fluid using positive electrospray ionization mode in presence of an internal standard, Icotinib. Ethyl acetate was used to extract Gefitinib from serum and cerebrospinal fluid samples and the linearity was observed over the concentration range 0.001-1.0 and 0.00005-0.050 mg/mL in human serum and

Cerebro spinal fluid respectively. Mass Zhao⁵ et al., developed a specific method for determination of Gefitinib in human plasma, mouse plasma and tissues using high performance liquid chromatography coupled to tandem spectrometry (LC-MS/MS) using electrospray positive ionization in presence of an internal standard, (d8)-Gefitinib. A mixture of acetonitrile and 0.1% formic acid (70:30) with 0.15 mL/min flow rate (Isocratic mode) with Waters X-Terra C₁₈ column was used for the study. The sample preparation involves protein precipitation and a very narrow range was reported 0.001-1.0, 0.005-1.0 and 0.005-1.0 mg/mL in human plasma, mouse plasma & tissues respectively. Nan Zheng⁶et al., developed a LC-MS-MS method for the quantitative estimation of Gefitinib and its major metabolites in tumor-bearing mouse plasmausing Dasatinib as internal standard. The sample was extracted by protein precipitation and the chromatographic study was performed using Acetonitrile: 0.1% formic acid as mobile phase on gradient mode with Agilent RRHD SB-C₁₈ column. The mass spectral analysis was performed using triple quadrupole mass spectrometry in positive multiple reaction monitoring mode.

Siva Kumar⁷ et al., developed a stability indicating HPLC method for Gefitinib for the separation and characterization of its related compounds using a mixture of 50 mM ammonium acetate (adjusted to pH 4.7 with trifluoroaceti acid) and acetonitrile as mobile phase on gradient mode with Inertsil C₈ column using PDA detection at 300 nm by preparing the samples in diluent consisting of 0.2% TFA: Acetonitrile (60: 40). The degradant products were characterized by ¹H NMR where dimethyl sulphoxide (DMSO-d6) was used as a solvent for solubilizing the impurities and MS/MS using electron spray ionization (ESI) detection on positive mode. Pramadvara and Mathrusri Annapurna⁸ developed a LC-ESI-Q-TOF/MS method for the separation, identification and quantification of 10 process related impurities and stress degradants of Gefitinib using LC MS Agilent 6120 single quadrupole with Agilent 1200 infinity series HPLC with Inertsil ODS 3V C₁₈ column. Mobile phase consisting of 10 mM ammonium acetate: acetonitrile (63:37, v/v) (pH was adjusted to 6.5 with glacial acetic acid) was used on isocratic mode (Detection wavelength 240 nm) was used for study.

Kiran Kumar⁹et al., and Gowri Sankar¹⁰et al., also developed HPLC methods for Gefitinib assay using dipotassium hydrogen ortho phosphate: methanol (10:90) (Detection at 246 nm; Rt 3.667 min) and 0.02M potassium di-hydrogen phosphate: acetonitrile (55:45) (Adjusted pH 6.5 with triethylamine) (Detection at 220 nm) respectively. Alagar Raja¹¹ et al., have developed a RP-HPLC method using 0.1% Triflouroacetic acid: Methanol (35:65) as mobile phase and Agilent column where Gefitinib was eluted at 3.4 min and the linearity range was reported as 5-30 mg/mL. Sandhya Mohan and Mangamma¹² have developed a HPLC method for the determination of Gefitinib in tablet dosage forms using methanol: dihydrogen potassium phosphate buffer (85:15) as mobile phase and Hypersil C₁₈ column with UV detection at 247 nm but a very narrow linearity range was reported i.e. 0.14-0.52 mg/mL. Navya Sree¹³ et al., have developed a RP-HPLC method for nanoformulation using acetonitrile and 40 mM ammonium formate buffer pH 2.5 (30:70) as mobile phase on HyperClone Phenomenex C₁₈ column with Phenomenex guard column where Gefitinib was eluted at 4.476 min and the linearity range was reported as 0.2-12 mg/mL. Ajay Singh¹⁴ et al., developed a HPLC method on gradient mode for the assay of Gefitinib using mobile phase, phosphate buffer (pH 3.5): acetonitrile and Inertsil ODS-2 C₁₈ column. Gefitinib was eluted at 6.98 min at 210 nm with linearity range 0.0015-0.07 mg/mL. Chandrashekara¹⁵ et al., have developed a HPLC method for the separation and estimation of five process-related impurities of Gefitinib using 130 mM ammonium acetate (Adjusted pH 5.0 with acetic acid) and acetonitrile (63:37) mixture as mobile phase and Inertsil ODS-3V column where Gefitinib was eluted at 6.777 min. Venkataramanna¹⁶ et al., have developed a rapid resolution liquid chromatography (RRLC) method for the identification of two potential impurities of Gefitinib using ammonium acetate and acetonitrile (40:60) mixture as mobile phase and Agilent XDB-C₁₈ column. Dudekula¹⁷ et al., have developed a stability indicating HPLC method for the determination of Gefitinib in bulk and its pharmaceutical formulations using acetonitrile: 0.5% ammonium dihydrogen phosphate buffer (30:70) mixture as mobile phase and YMCODS-AQ column. Gefitinib was eluted at 7.43 min at 205 nm with linearity range 0.05-0.15 mg/mL. Sreedevi¹⁸ et al., have developed a stability indicating HPLC method for the quantification of Gefitinib using mobile phase mixture, phosphate buffer (pH 3.6): acetonitrile (55:45) and Hypersil BDS C₁₈ column. Gefitinib was eluted at 4.179 ± 0.2 min at 248 nm with linearity range 0.25-0.15 mg/mL. In the present study the authors have developed a new stability indicating RP-HPLC method for the estimation of Gefitinib and the method was validated as per ICH guidelines.

Figure 1: Structure of Gefitinib

MATERIAL AND METHODS:

Gefitinib samples (>99.5 purity) obtained as gift samples from Therdose Pvt Ltd (Hyderabad, India). HPLC grade methanol was obtained from Merck (India) and other chemicals such as tetra butyl ammonium hydrogen sulphate (TBHS), sodium hydroxide, hydrochloric acid and hydrogen peroxide (30% w/v) were procured from Qualigens (India) and S.D. Fine Chemicals (India). HPLC grade water was obtained from Milli-Q Gradient Millipore system.

Preparation of 10 mM Tetra butyl ammonium hydrogen sulphate solution (pH 3.4)

Tetra butyl ammonium hydrogen sulphate (C₁₆H₃₇NO₄S) is an ion pairing agent. The molecular weight of tetra butyl ammonium hydrogen sulphate is 339.54 grams/mole. About 3.3954 grams Tetra butyl ammonium hydrogen sulphate was accurately weighed and transferred to a 1000 mL volumetric flask and dissolved in HPLC grade water to prepare 10 mM solution and the pH of the resulting solution is 3.4. This buffer solution was filtered through 0.42 micron membrane filter and used for the preparation of mobile phase.

Preparation of mobile phase and diluent:

The mobile phase was prepared using tetra butyl ammonium hydrogen sulphate buffer and methanol in 50: 50, v/v ratio. Both organic and aqueous phase were sonicated and filtered prior to use. The diluent was prepared by mixing tetra butyl ammonium hydrogen sulphate buffer and methanol in 60: 40, v/v ratio.

Preparation of stock solution of Gefitinib:

10 mg of Gefitinib was accurately weighed and dissolved in 10 mL volumetric flask in methanol and the volume was made up volume. This solution is known as stock solution (1000 µg/mL) and this was further diluted with mobile phase to produce a concentration of 100 µg/mL which is known as working standard solution and these solutions were stored in refrigerator at 2-8ºC.

Instrumentation and Chromatographic conditions

Shimadzu Model CBM-20A/20 Alite HPLC system with PDA detector and Agilent C₁₈ column were used for the chromatographic study. Mobile phase mixture consisting of Tetra butyl ammonium hydrogen sulphate and Methanol in the ratio 50:50, v/v with a flow rate 0.8 mL/min was chosen for the chromatographic elution of Gefitinib (Detection wavelength 340 nm). The injection volume was 20 µL and the total run time was 10 minutes.

Method validation¹⁹

Linearity study

A series of 0.1-80 µg/mL of Gefitinib solutions were prepared from the stock solution on dilution with the diluent and each of these solutions were injected (n=3) into the HPLC system and the chromatograms were recorded. The peak area for each of these solutions (n=3) was noted at its retention time and the mean peak area was calculated. Calibration curve was drawn by plotting the concentration of Gefitinib solutions on the x-axis and the corresponding mean peak area on the y-axis. The LOD and LOQ were calculated from the signal to noise ratio (S/N). The LOD is 3.3 times the signal to noise ratio and that of LOQ is 10 times the signal to noise ratio.

Precision study:

Precision of the method was evaluated intra-day and inter-day precision studies. Three different concentration solutions (5, 10 and 20 µg/mL) of Gefitinib were prepared within the linearity range on the same day (intra-day precision) and on three consecutive days (inter-day precision) and the chromatographic study was performed. The mean peak area (n=3) and thereby the % RSD was calculated.

Accuracy study

Accuracy of the method was measured by spiking the drug formulation (20 µg/mL) solution (50, 100, 150%) with a known concentration of standard drug (n=3) where the final concentrations were found to be 30, 40 and 50 µg/mL. The mean peak area was calculated from the chromatograms obtained and finally the % RSD was calculated from the linear regression equation.

Robustness study:

The robustness of the method was proved by incorporating a very small changes in the optimized chromatographic conditions such as pH (±0.1; 3.3 and 3.5), mobile phase composition (± 2%; 48:52 and 52:48), flow rate (± 0.1 mL; 0.7 and 0.9 mL/min) and detection wavelength (± 2 nm; 342 and 338 nm).

Assay of Gefitinib tablets:

Gefitinib is available as film coated tablets (Label claim: 250 mg) with brand names Geftinat (Natco Pharma Limited), Gefticip (Cipla Ltd), Grexam (SUN Pharmaceutical Industries Ltd) and Iressa (Astra Zeneca). Twenty tablets of Gefitinib were accurately weighed and an amount of powder equivalent to 100 mg of Gefitinib from two different brands was accurately weighed and transferred into two different 100 mL volumetric flasks. HPLC grade methanol was added, sonicated and filtered. The resulting mixture was then diluted with the diluent (TBHS: Methanol 60: 40, v/v). 20 µL of each of these two different branded solutions were then injected into the system thrice and the average peak area was calculated from the resultant chromatograms. The assay was performed and the amount of Gefitinib was calculated with the help of calibration curve.

Stress degradation studies²⁰

Stress degradation studies were performed to determine the stability of Gefitinib towards stress conditions such as acidic hydrolysis, basic hydrolysis, oxidation and thermal degradation. The specificity of the method can be known from the stability studies and therefore Gefitinib was exposed to the following stress conditions and the stability was studied. Acidic degradation was performed by heating Gefitinib solution with 1 mL of 0.1 N HCl solution at 80°C for 30 minutes on a water bath. The stressed sample was then cooled, neutralized with 1.0 mL 0.1N sodium hydroxide solution, diluted with mobile phase and then 20 µL of the solution was injected into the HPLC system. Alkaline degradation was performed by heating Gefitinib solution with 1.0 mL 0.1N sodium hydroxide solution at 80^°C for 30 minutes on a water bath. The stressed sample was then cooled, neutralized with 1.0 mL of 0.1 N HCl solution, diluted with mobile phase and then 20 µL of the resulting solution was injected into the HPLC system. Thermal degradation was performed by heating the Gefitinib solution at 80°C for 30 minutes on a water bath and then cooled, diluted with mobile phase and 20 μL of the resulting solution was injected in to the HPLC system. Oxidative degradation was performed by heating Gefitinib solution with 1.0 mL 30% hydrogen peroxide solution at 80°C for 30 minutes on a water bath. The stressed sample was then cooled, diluted with mobile phase and then 20 µL of the resulting solution was injected into the HPLC system.

RESULTS AND DISCUSSION:

The authors have developed a new stability indicating RP-HPLC method for the determination of Gefitinib in API and tablets. Ion pair chromatography is a widely used analytical technique especially for performing reverse phase chromatography. In the present study ion pairing reagent, tetra butyl ammonium hydrogen sulphate was chosen for the aqueous phase of the mobile phase. Earlier different authors have proposed various analytical methods for Gefitinib and the present proposed method was compared with the previously published methods in Table 1.

Table. 1. Literature survey of Gefitinib

Mobile phase (v/v)	λ (nm)	Linearity (mg/mL)	Method	Reference
Acetonitrile: water (50:50) (Human serum, Cerebrospinal fluid)	-	-	LC/MS/MS	4
Acetonitrile: 0.1% Formic acid (70:30) (Human plasma, Mouse plasma & Tissues)	-	0.001-1.0 0.005-1.0 0.005-1.0	LC/MS/MS (Isocratic mode)	5
Acetonitrile: 0.1% Formic acid (Mouse plasma)	-	-	LC-MS/MS (Gradient mode)	6
50mM Ammonium acetate: Acetonitrile	300	-	LC-MS/MS & NMR (Gradient mode)	7
10 mM Ammonium acetate: Acetonitrile (63: 37%) (pH adjusted to 6.5 with acetic acid)	240	0.2 - 750	LC-ESI-Q-TOF/MS (Isocratic mode) 10 Impurities Stability indicating	8
Dipotassium Hydrogen ortho phosphate: Methanol (10:90)	246	25-300	HPLC	9
0.02M Potassium di-hydrogen phosphate: Acetonitrile (55:45) (Adjusted pH 6.5 with tri ethyl amine)	220	10-60	HPLC	10
0.1% Triflouroacetic acid: Methanol (35:65)	246	5-30	HPLC	11
Methanol: Dihydrogen potassium phosphate (85:15)	247	0.14 - 0.52	HPLC	12
Acetonitrile: 40 mM Ammonium formate buffer (pH 2.5) (30: 70)	248	0.2-12	HPLC Nano formulation	13
Acetonitrile: phosphate buffer (pH 3.5)	210	1.5-0.70	HPLC (Gradient mode)	14
Acetonitrile: 130 mM Ammonium acetate and (37: 63); pH 5.0 (Adjusted pH 5.0 with acetic acid)	260	0.1-2.0 (Impurities) 25-500 (Gefitinib)	HPLC (5 Impurities)	15
Ammonium acetate: Acetonitrile (40:60)	250	-	RRLC (2 Impurities) (Gradient mode)	16
Acetonitrile: 0.5% Ammonium dihydrogen phosphate buffer (30:70)	205	50-150	HPLC Stability indicating	17
Acetonitrile: Phosphate buffer(55:45) (pH 3.6)	248	25-150	HPLC Stability indicating	18
10 mM Tetra butyl ammonium hydrogen sulphate: Methanol (50: 50) (pH 3.4) (Isocratic mode)	340	0.1-80	HPLC Ion pairing agent Stability indicating	Present method

Trial 1: Mobile phase: TBHS: Methanol (60:40); Flow rate: 0.6 mL/min; Rt 7.383 min

Theoretical plates: 6039; Tailing factor: 2.119 (>2)

Trial 2: Mobile phase: TBHS: Methanol (60:40); Flow rate: 0.8 mL/min; Rt 3.994 min

Theoretical plates: 1993 (<2000); Tailing factor: 1.343

Trial 3: Mobile phase: TBHS: Methanol (50:50); Flow rate: 0.8 mL/min; Rt 3.422 min

Theoretical plates: 5629; Tailing factor: 1.221 (Method optimized)

Figure 2: Representative chromatograms of Gefitinib during method optimization

Table 2: Method optimization of Gefitinib

Trial	Column	Mobile phase (v/v)	Flow rate (mL/min)	Rt (min)	Theoretical plates	Tailing factor	Observations
1	Agilent C₁₈	60: 40	0.6	7.383	6039	2.119	Rt is more Peak tailing
2	Agilent C₁₈	60: 40	0.8	3.994	1993	1.343	Rt is more
3	Agilent C₁₈	50:50	0.8	3.422	5629	1.221	Method optimized

Method validation:

Gefitinib obeys Beer-Lambert’s law over the concentration range of 0.1-80 µg/mL (% RSD 0.25-0.61) (Table 3) and representative chromatograms of the placebo and Gefitinib API were shown in Figure 3A and Figure 3B. The LOD and LOQ were found to be 0.2931 mg/mL and 0.8947 mg/mL respectively. The linear regression equation was found to be y = 70782x + 6171.6 with correlation coefficient 0.9999 and the calibration curve was shown in Figure 4. The % RSD was found to be 0.23-0.42 (Intraday) (Table 4) and 0.51-1.05 (Inter-day) (Table 5) in precision studies which is less than 2.0 indicating that the method is precise. The % recovery in accuracy studies was found to be 99.37-99.83% (Table 6) and % RSD was (0.68-1.08) less than 2% indicating that the method is accurate. The % RSD in robustness study was found to be 0.43-1.39 which was less than 2% indicating that the method is robust (Table 7).

Table 3: Linearity study of Gefitinib

Conc. (µg/mL)	*Mean peak area	% RSD
0	0	-
0.1	7682	0.51
0.2	15423	0.36
0.5	37263	0.25
1	75693	0.31
2	141264	0.29
5	364255	0.27
10	759071	0.61
20	1411117	0.52
40	2823659	0.43
80	5672604	0.56

*Mean of three replicates

Figure 4: Calibration curve of Gefitinib

Table 4: Intraday precision study of Gefitinib

Conc. (µg/mL)	*Mean peak area	Statistical Analysis
Conc. (µg/mL)	*Mean peak area	*Mean ± SD (% RSD)
5	364255	364286.00 ± 837.86 (0.23)
5	364291
5	364312
10	759071	759045.33 ± 3187.99 (0.42)
10	758962
10	759103
20	1411117	1411148.33 ± 4092.33 (0.29)
20	1411196
20	1411132

*Mean of three replicates

Table 5: Inter day precision study of Gefitinib

Conc. (µg/ mL)	*Mean peak area			*Mean ± SD (% RSD)
Conc. (µg/ mL)	Day 1	Day 2	Day 3	*Mean ± SD (% RSD)
5	364255	365162	366804	365407 ± 1863.58 (0.51)
10	759071	758619	759153	758947.67 ± 6375.16 (0.84)
20	14111132	14112031	14111998	14111720.33 ± 148173.06 (1.05)

*Mean of three replicates

Table 6: Accuracy study of Gefitinib

Spiked

Conc. (μg/mL)

Formulation

(μg/mL)

Total Conc.

(μg/mL)

*Conc. obtained (μg/mL)

± SD (%RSD)

Recovery

10 (50%)

29.81 ± 0.2027 (0.68)

99.37

20 (100%)

39.93 ± 0.3674 (0.92)

99.83

30 (150%)

49.89 ± 0.5388 (1.08)

99.78

*Mean of three replicates

Table 7: Robustness study of Gefitinib (10 μg/mL)

Parameter	Condition	*Mean peak area ± SD (% RSD)
Flow rate (± 0.1 mL/min)	0.7	759221 ± 8275.51 (1.09)
	0.8
	0.9
Detection wavelength (± 2 nm)	338	759124 ± 3264.23 (0.43)
	340
	342
Mobile phase composition Tetra butyl ammonium hydrogen sulphate: Methanol (± 2 %, v/v)	48:52	759193 ± 5162.51 (0.68)
	50:50
	52:48
pH	3.5	759298 ± 10554.24 (1.39)
	3.4
	3.3

*Mean of three replicates

Assay of Gefitinib tablets:

The assay of Gefitinib was found to be 99.72-99.84 (Table 8) in tablets. The chromatogram observed for one of the brands of Gefitinib tablet formulation and that of the placebo were shown in Figure 3C. The excipients of the formulation have not interfered with the pure drug peak.

Table 8: Assay of Gefitinib tablets

S. No.	Brand name	Label claim (mg)	*Observed amount (%w/w)	% Recovery*
1	Brand I	250	249.61	99.84
2	Brand II	250	249.29	99.72

*Mean of three replicates

Stress degradation studies of Gefitinib:

Gefitinib (API) was eluted as a sharp peak at 3.422 min with accepted system suitability parameters i.e. theoretical plates 5526.086 (> 2000) and tailing factor 1.306 (< 1.5). During the acidic degradation studies Gefitinib has undergone 18.54% degradation with theoretical plates 4855.141 (> 2000) and tailing factor 1.229. During the alkaline degradation studies Gefitinib has undergone 13.38% degradation with theoretical plates 5705.993 (> 2000) and tailing factor 1.317. During the oxidative degradation studies Gefitinib has shown 9.11% degradation with theoretical plates 5814.70 (> 2000) and tailing factor 1.300 with an extra peak at 2.294 min with resolution 6.537 which is greater than 2. During the thermal degradation Gefitinib has shown 11.83% degradation with theoretical plates 5663.189 (> 2000) and tailing factor 1.306. The method is highly specific as the Gefitinib drug peak has not at all interfering with any other degradant peak. The % recovery in all the degradation studies was less than 15% (Table 9) and the corresponding chromatograms obtained during the stress degradation studies were shown in Figure 5.

Table 9: Stress degradation studies of Gefitinib

Stress condition (Temp °C/ Time min)	Rt (min)	*Mean peak area	% Recovery	% Drug degradation	Theoretical plates	Tailing factor
Standard drug	3.422	759071	100	-----	5526.086	1.306
Acidic degradation 0.1N HCl /80°C/30 min	3.497	618319	81.46	18.54	4855.141	1.229
Alkaline degradation 0.1N NaOH/80°C/30 min	3.415	657538	86.62	13.38	5705.993	1.317
Oxidative degradation 30% H₂O₂/ 80°C/30 min	3.380 2.294	689909	90.89	9.11	5814.70 3512.658	1.300 1.314
Thermal degradation 80°C/30 min	3.409	669294	88.17	11.83	5663.189	1.306

*Mean of three replicates


Acid degradation	Alkaline degradation

Oxidative degradation	Thermal degradation
Figure 5: Chromatograms of Gefitinib during stress degradation studies

CONCLUSION:

A new stability indicating RP-HPLC was developed for the determination of Gefitinib and validated as per ICH guidelines. The method is specific and no degradants were interfering with Gefitinib peak and there is no interference of excipients used in the tablet formulation. Gefitinib is quite stable towards all degradations as the percentage of degradation is less than 20%. The proposed method is simple precise, accurate and robust and is suitable for the pharmacokinetic studies.

ACKNOWLEDGEMENT:

The authors are grateful to Therdose Pvt Ltd (India) for providing the gift samples of Gefitinib. The authors have no conflict of interest.

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Received on 21.09.2021 Modified on 25.10.2021

Research J. Pharm. and Tech 2021; 14(10):5449-5456.

DOI: 10.52711/0974-360X.2021.00950