Analysis of Gefitinib listed in the Japanese Pharmacopoeia 18th Edition

Introduction

Gefitinib, newly listed in the 18th edition of the Japanese Pharmacopoeia (JP18), is a therapeutic agent used in the treatment of non-small cell lung cancer (NSCLC), particularly in patients for whom surgery intervention is not feasible. It functions as a selective inhibitor of mutant epidermal growth factor receptor (EGFR) tyrosine kinase, thereby suppressing tumor cell proliferation and disease progression. In pharmaceutical quality control, accurate and reliable determination of drug purity and assay content is essential to ensure compliance with regulatory standards. The JP18 specifies analytical requirements for the evaluation of gefitinib, including system suitability criteria and method performance characteristics. This application note describes the HPLC methods for the purity and assay of gefitinib in accordance with JP18 guidelines, with a focus on system suitability evaluation, method precision, and method reproducibility to demonstrate the robustness and reliability of the analytical approach.

Experimental

Instruments

Pump: PU-4580

Degassing unit: DG-4580

Autosampler: AS-4550

Column oven: CO-4060

UV detector: UV-4570

LC Conditions

Column: Unifinepak C18 (3.0 mm I.D. x 100 mm L, 3 µm)

Eluent: Ammonium acetate solution (Dissolve 3.0 g ammonium acetate with water and fill up to 310 mL)/acetonitrile (31/19)

Flow rate: 0.9 mL/min

Column temp: 60 ºC

Wavelength: 247 nm

Injection volume: 5 µL

Standard: Gefitinib (The dissolving and diluting solvent is a mixture of 0.2 % trifluoroacetic acid aqueous solution/acetonitrile (3/2))

Keywords

Japanese Pharmacopoeia, gefitinib, UV detector, system suitability

Results

An overview of the system suitability for purity and assay tests on gefitinib is shown in Table 1.

Table 1. Overview of purity and assay system suitability tests for gefitinib

	Test	Criteria
Purity	Detectability	When the purity standard solution (0.35 mg/L) is diluted to a concentration of 0.175 mg/L and the diluted purity standard (0.175 mg/L ) is injected under the above operating conditions, the signal-to-noise ratio for the gefitinib peak is ≥10.
Purity	System Performance	Follow the procedure for the system performance test for the assay.
Purity	System Reproducibility	When 5 µL of the purity standard solution is injected six times under the above operating conditions using 5 µL injections, the relative standard deviation of the gefitinib peak area is ≤2.0%.
Assay	System Performance	Dissolve 15 mg of 3,4-dichloroaniline in 60 mL of the assay standard solution (350 mg/L). When 5 µL of the assay standard solution (350 mg/L) is injected under the above operating conditions, 3,4-dichloroaniline and gefitinib are eluted in this order, and the resolution between the peaks is ≥5.
Assay	System Reproducibility	When 5 µL of the assay standard solution is injected six times under the above operating conditions, the relative standard deviation of the gefitinib peak area is ≤1.0%.

(1) Purity

Figure 2 shows the results of the purity detectability test. The signal-to-noise ratio of the bicalutamide peak for the diluted standard solution (0.175 mg/L) was 50.7, which met the specified criterion of ≥10.

Fig. 2 Chromatogram of gefitinib for detectability purity test (0.175 mg/L)

Figure 3 shows the chromatograms of the gefitinib standard solutions (n = 6), while Table 2 summarizes the peak area reproducibility results obtained from the system reproducibility test for the purity analysis. The relative standard deviation of the peak area was 0.26%, satisfying the acceptance criterion of ≤2.0%.

Fig. 3 Chromatogram of gefitinib standard solution (0.35 mg/L, n = 6)

Table 2. Gefitinib peak area reproducibility (n = 6)

Injection Number	Peak Area
1	5,097
2	5,119
3	5,103
4	5,079
5	5,093
6	5,097
Average	5,098
SD	13
RSD [%]	0.26

(2) Assay

Figure 4 shows the chromatogram of a standard solution containing 3,4-dichloroaniline

(250 mg/L) and gefitinib (350 mg/L). The compounds eluted in the expected order, with

3,4-dichloroaniline preceding gefitinib. The measured resolution between the two peaks

was 9.0, satisfying the JP18 acceptance criterion of ≥5.

Fig. 4 Chromatogram of 3,4-dichloroaniline + gefitinib (250 mg/L and 350 mg/L, respectively)

Figure 5 shows the chromatograms of the assay standard solution (n = 6), while Table 3 summarizes the corresponding system suitability results. The relative standard deviation of the gefitinib peak area was 0.11%, satisfying the acceptance criterion of ≤1.0%.

Fig. 5 Chromatogram of gefitinib standard solution (350 mg/L, n = 6)

Table 3. Gefitinib peak area reproducibility (n = 6)

Injection Number	Peak Area
1	4,922,389
2	4,932,506
3	4,937,250
4	4,926,834
5	4,932,461
6	4,930,941
Average	4,930,397
SD	5,153
RSD [%]	0.11

Conclusion

System suitability testing for both the purity and assay methods of gefitinib, as specified in the 18th edition of the Japanese Pharmacopoeia, was successfully performed. As summarized in Table 4, all evaluated parameters met the specified acceptance criteria of the Japanese Pharmacopoeia.

Table 4. System suitability criterion and results for the purity and assay tests of gefitinib

	Test	Test Item	Criterion	Result	Judgement
Purity	Detectability	Signal-to-noise ratio	≥ 10	50.7	Passed
Purity	System Reproducibility	Relative standard deviation of peak area	≤ 2.0 %	0.26 %	Passed
Assay	System Performance	Resolution	≥ 5	9.0	Passed
Assay	System Reproducibility	Relative standard deviation of peak area	≤ 1.0 %	0.11 %	Passed

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