MS and its application to a pharmacokinetics study

Journal of Chromatography B, 1011 (2016) 215–222

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Journal of Chromatography B journal homepage: www.elsevier.com/locate/chromb

Simultaneous determination of gefitinib and its major metabolites in mouse plasma by HPLC–MS/MS and its application to a pharmacokinetics study Nan Zheng a,b,1 , Can Zhao a,c,1 , Xi-Ran He a,c , Shan-Tong Jiang a,c , Shu-Yan Han a,c,∗ , Guo-Bing Xu a,b , Ping-Ping Li a,c,∗ a

Key laboratory of Carcinogenesis and Translational Research (Ministry of Education), Beijing 100142, PR China National Drug Clinical Trial Center, Peking University Cancer Hospital & Institute, Beijing 100142, PR China c Department of Integration of Chinese and Western Medicine, Peking University Cancer Hospital & Institute, Beijing 100142, PR China b

a r t i c l e

i n f o

Article history: Received 28 October 2015 Received in revised form 8 December 2015 Accepted 5 January 2016 Available online 8 January 2016 Keywords: Gefitinib Metabolites LC–MS/MS Mouse plasma Pharmacokinetics

a b s t r a c t Gefitinib (Iressa) is the first oral EGFR tyrosine kinase inhibitor and it brings benefits to non-small cell lung cancer patients with EGFR mutation. In this study, a simple, rapid and credible high performance liquid chromatography–tandem mass spectrometry method was established and validated for the simultaneous quantification of gefitinib and its main metabolites M523595, M537194, M387783 and M608236 in NSCLC tumor-bearing mouse plasma. Sample extraction was done by protein precipitation using acetonitrile containing dasatinib as the internal standard. The chromatography run time was 6 min using an Agilent RRHD SB-C18 column with a gradient of acetonitrile and water (0.1% formic acid, v/v). The mass analysis was performed by a triple quadrupole mass spectrometry in positive multiple reaction monitoring mode. The calibration range was 0.5–100 ng/mL for M608236 and 1–200 ng/mL for other analytes with the correlation coefficients (r2 ) ≥ 0.99. For quality control samples, inter- and intra-assay precision was less than 15% and accuracies ranged from 92.6% to 107.58% for all analytes. The extraction recoveries were in the range of 86–105% and no significant matrix effect was observed. This simple and reproducible highthroughput method was successfully applied to the pharmacokinetic study of gefitinib and its major metabolites in mouse. © 2016 Elsevier B.V. All rights reserved.

1. Introduction Gefitinib (Iressa® ) is an anilinoquinazoline compound with the chemical name 4-quinazolinamine, N-(3-chloro-4-flurophenyl)-7methoxy-6-[3-(4-morpholinyl)propoxy]. It is an orally active and a

Abbreviations: TKI, tyrosine kinase inhibitor; NSCLC, non-small cell lung cancer; LC–MS/MS, liquid chromatography–tandem mass spectrometry; EGFR, epidermal growth factor receptor; MRM, multiple reaction monitoring; CYP, cytochrome P450; PK, pharmacokinetic; ESI, electrospray ionization; QC, quality control; IS, internal standard; LLOQ, lower limit of quantification; CE, collision energy; S/N-ratio, signal to noise ratio; RSD, relative standard deviation; RE, relative errors; ULOQ, upper limit of quantification; UHQC, ultra-high quality control; t1/2 , half-time; Tmax , peak time; AUC, area under concentration–time curve; Cmax , the peak concentration. ∗ Corresponding authors at: Department of Integration of Chinese and Western Medicine, Peking University School of Oncology, No. 52 Fucheng Road, Haidian District, Beijing 100142, PR China. Fax: +86 10 88196069. E-mail addresses: [email protected] (S.-Y. Han), [email protected] (P.-P. Li). 1 These authors contributed equally to this work. http://dx.doi.org/10.1016/j.jchromb.2016.01.006 1570-0232/© 2016 Elsevier B.V. All rights reserved.

selective small-molecule epidermal growth factor receptor (EGFR) TKI indicated for the treatment of locally advanced or metastatic NSCLC with sensitive mutations of EGFR [1,2]. Many clinical studies have confirmed that NSCLC patients with EGFR-active gene mutations could get benefits from gefitinib treatment [3,4]. Gefitinib is extensively metabolized by cytochrome P-450 (CYP) 3A4 and 2D6 [5]. Oxidation of the morpholine ring, oxidative defluorination and O-demethylation of the methoxy-substituent on quinazoline nucleus represent the main routes of gefitinib metabolism [5,6]. CYP3A4 catalyzed gefitinib to produce metabolites of M537194, M608236 and M387783, while CYP2D6 exerted rapid and extensive metabolism of gefitinib to M523595 [6]. Monitoring gefitinib and its metabolites may help to minimize the risk of drug–drug interactions and dose-related toxicity due to its extensive hepatic metabolism. Therefore, an analytical method is required to determine the concentrations of gefitinib and its major metabolites as well as their pharmacokinetic (PK) profiles. To our knowledge, there are some validated methods for quantification of gefitinib and M523595 (Table 1), but other metabolites

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Table 1 Published methods for quantification of gefitinib using liquid chromatography–tandem mass spectrometry. Compound

Matrix

Sensitivity (LOQ)

Sample extraction

Run time

Mobile phase (v/v)

Gefitinib [7] Gefitinib [8]

0.50 ng/mL 1 ng/mL (human); 5 ng/mL (mouse)

LLE PP

4 min 3 min

70% acetonitrile:30% formic acid (1%) 70% acetonitrile:30% water containing 0.1% formic acid

Gefitinib [9]

Human plasma Human plasma; mouse plamsa and tissues Human plasma

0.1 ng/mL

LLE

10 min

Gefitinib [10]

Human plasma

0.5

LLE

5 min

Gefitinib, M523595 [11] Gefitinib & metabolites [16]

Human plasma

5 ng/mL

LLE

3 min

Rat plasma; dog plamsa

PP

–

Three TKIs including gefitinib [12]

Human plasma

5 ng/mL (M523595, M537194); 2 ng/mL for others 5 ng/mL

80% acetonitrile:20% aqueous ammonium acetate (1% w/v) 80% acetonitrile:20% aqueous ammonium acetate (1% w/v) 30% acetonitrile:70% water containing 0.1% formic acid 80% acetonitrile:20% aqueous ammonium acetate (1% w/v)

LLE

–

Nine TKIs including gefitinib [13] Nine TKIs including gefitinib [14]

Human plasma

0.40 ng/mL

SPE

4 min

Human plasma

20 ng/mL

PP

10 min

Four TKIs including gefitinib [15]

Human plasma and cell culture

11.2 nM

PP

5 min

65% water:35% methanol containing 0.01% formic acid and 2 mM ammonium acetate 90% acetonitrile:10% 4 mM ammonium formate 20% ammonium hydroxide in water 10 mM:80% ammonium hydroxide in methanol 1 mM 66.6% acetonitrile:25% 20 mM ammonium acetate:8.3% methanol

LOQ: limit of quantification; LLE: liquid–liquid extraction; PP: protein precipitation; SPE: solid phase extraction.

Table 2 Summary of the MRM transitions for gefitinib, its metabolites and internal standard used in the LC–MS/MS analysis. Compound

Transitions (Da)

Dwell time (ms)

Fragment-or (V)

Collision energy (V)

Mean retention time (min)

Quantifier ions Gefitinib M523595 M537194 M387783 M608236 Dasatinib(IS)

447 → 128 433 → 128 421 → 320 445 → 128 449 →n130 488 → 401

40 40 40 40 40 40

150 150 150 150 150 190

28 23 22 24 24 31

1.86 1.82 1.80 1.46 2.28 2.32

Qualifier ions Gefitinib M523595 M537194 M387783 M608236

447 → 100.1 433 → 100.1 421 → 102.1 445 → 100.1 449 → 320

40 40 40 40 40

150 150 150 150 150

28 25 18 35 28

1.86 1.82 1.80 1.46 2.28

of gefitinib were not involved [7–15]. McKillop et al. reported a method for analyzing gefitinib and its metabolites, but the methodology and validation was not studied, and the analytes were with unsatisfactory purity (ranging from 44.7% to 89.8%) [16]. As mentioned above, there are a number of researches involving the PK behavior of gefitinib, but lack of assays for PK profiles of its metabolites. In this study, we developed a simple, rapid and accurate LC–MS/MS method to simultaneously determine gefitinib, M523595, M537194, M387783 and M608236 in mouse plasma. We applied this established method to study the PK profiles of gefitinib and its major metabolites in tumor-bearing mouse after a single oral administration of gefitinib.

2. Materials and methods 2.1. Chemicals and materials Gefitinib was purchased from AstraZeneca (Cheshire, UK), and dasatinib was produced from Bristol-Myers Squibb (NY, USA). M537194, M523595, M387783, M608236 were synthesized in Yaosu Technology Ltd. (Purity ≥ 98%, Beijng, China). HPLC grade methanol and acetonitrile were from Merck (Darmstadt, Germany). Analytical standard formic acid was from Sigma–Aldrich

(98% purity, mass spectrometry grade, St. Louis, Missouri, USA). Chromatographic pure water was produced by Milli-Q® Water Purification Systems (Merck Millipore, MA, USA). The other chemicals used in this study were all of chromatographic grade.

2.2. Instrumentations and chromatographic conditions The LC–MS/MS method was performed on an Agilent 1290 Infinity LC system (Agilent Technologies, USA), which mainly consisted of a G4220A binary pump, a G4226A infinity auto-sampler, a G1316C column heater and a G6460A triple-quadrupole mass spectrometer with an electrospray ionization (ESI) source. The separation was achieved on an Agilent RRHD SB-C18 column (2.1 mm × 50 mm, 1.8 ␮m, Agilent Technologies, USA). An aliquot of 2.0 ␮L of the sample was injected on the column. Mobile phase was a mixture of 0.1% (v/v) formic acid in water (A) and acetonitrile (B). The gradient grogram of mobile phase was as follows: 10% B at 0–0.5 min; 10–50% B at 0.5–2.5 min; 50–95% B at 2.5–3 min; held 95% B at 3–4.5 min; 95–10% B at 4.5–4.6 min, 10% B for equilibration of the column. The total analytical run time was 6.0 min per sample, including equilibration time. The flow rate was 0.4 mL/min and the column was maintained at 40 ◦ C. The quantifier- and qualifier-ions for gefitinib and its metabolites are also tabulated in Table 2.

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Fig. 1. The chemical structures and MS/MS spectra of gefitinib, M523595, M608236, M537194, M387783 and dasatinib (IS).

2.3. Standard and sample preparation

2.4. Method validation

2.3.1. Preparation of stock solutions For all compounds, sets of stock solutions were prepared from two independent weightings: one is for the calibration standards and another is for the quality control (QC) samples. The stock solutions were prepared by dissolving gefitinib, M523595, M537194, M387783, M608236 and dasatinib in methanol to be 1.0 mg/mL, respectively. Dasatinib was served as internal standard (IS) and dissolved in methanol to get a 10 ␮g/mL working solution [17]. All the solutions were stored at 4 ◦ C in the dark and brought to room temperature before use.

In accordance with the FDA bioanalytical method validation guide [18], the LC–MS/MS method was validated in terms of specificity, linearity, precision, accuracy, matrix effect, extraction recovery, stability, dilution integrity and carry-over. The specificity was evaluated by analyzing the blank biological samples, blank biological matrix samples spiked with IS and samples at lower limit of quantification (LLOQ), respectively. Calibration curves were established by plotting the peak area ratios of the analysts to IS (Y-axis) versus the nominal concentration of the compounds (X-axis) through weighted least-squares linear regression analysis with a weighting factor of 1/x. The LLOQ was defined as the lowest concentration on the calibration curve, at which precision and accuracy in five replicates should not exceed 20%. Deviations of the back-calculated concentrations of standard samples from their theoretical values were set within ±15% for all other calibration levels. The accuracy and precision were assessed by QC samples with five replicates (low, middle and high concentration) on three continuous days. Relative standard deviation (RSD%) was used to evaluate the intra- and inter-day precision and it should not exceed 15%. To assess the accuracy, the relative error (RE%) was calculated according to the formula: RE% = [(assayed value − nominal value)/nominal value] × 100%. The extraction recovery and matrix effect of gefitinib and its metabolites were determined using QC samples at three concentrations. The extraction recovery was obtained from the peak response ratio of blank plasma spiked with standard followed by extraction and working solution at the same concentration in six replicates (representing 100% recovery). The matrix effect was evaluated by comparing the peak area of the standards spiking in the extracted blank plasma with working solution dissolved in mobile phase (33% acetonitrile, v/v) at the equivalent concentration (three replicates).

2.3.2. Preparation of calibration standards and QC samples For preparation of the calibration standards, the stock of each compound at 1 mg/mL was diluted with methanol to obtain a mixed standard solution that containing 10 ␮g/mL M608236 and 20 ␮g/mL other components. The standard samples mixture was diluted with mouse blank plasma to achieve a final concentration at 100, 75, 50, 25, 12.5, 5, 2.5 and 0.5 ng/mL for M608236, while 200, 150, 100, 50, 25, 10, 5 and 1 ng/mL for other compounds. In the same way, the QC samples were prepared with blank plasma at 1.5, 12.5 and 80 ng/mL for M608236, and the remaining compounds were set at 3, 25 and 160 ng/mL. 2.3.3. Sample preparation A simple protein precipitation method was carried out to extract gefitinib and its metabolites from QC samples, calibration standards, and all the plasma samples. The IS was diluted with acetonitrile to a final concentration of 30 ng/mL, added to each sample and vortexed thoroughly to let protein sedimentation. After centrifugation at 14,000 rpm for 10 min at 4 ◦ C, the supernatant was obtained for LC–MS/MS analysis (total dilution factor 3). The final concentration of IS in all the samples was 20 ng/mL.

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Fig. 2. Representative chromatograms of blank plasma from mouse (A) and blank mouse plasma spiked with gefitinib, the metabolites and dasatinib (IS) (B) at LLOQ level.

The stabilities of gefitinb and its metabolites in mouse plasma were investigated under specific conditions as follows: (1) room temperature for 6 h (bench-top stability); (2) 10 ◦ C for 24 h (autosampler stability) (3) long-term stability (−80 ◦ C for 30 days); (4) three freeze–thaw cycles (freeze–thaw stability). Samples were tested with six replicates. It is regarded as stable if the average percentage concentration deviation was within 15% of the actual value. Carry-over was tested by injecting two processed blank matrix samples sequentially after injecting an upper limit of quantification (ULOQ) sample [19]. Carry-over should not exceed 20% of the LLOQ and 5% of the IS in the blank samples. In order to ensure the

analysis of samples over ULOQ, the dilution integrity was carried out by diluting an ultra-high QC (UHQC) samples at the five times of the highest calibration level. Six replicates of UHQC samples were diluted 10- and 20-fold with blank mouse plasma and analyzed. The acceptance criterion was that the accuracy and precision of diluted UHQC samples also should be within ±15%. 2.5. Pharmacokinetics study The animal experimental procedures were performed according to National Guidelines on the Proper Care and Use of Animals in Laboratory Research. Fifty-five female nude mice (18–20 g) were

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Table 3 Calibration curve, correlation coefficients and linear ranges of blank mouse plasma spiked with gefitinib and its main metabolites. Compound

Range (ng/mL)

Calibration curve

Correlation coefficient (r2 )

LLOQ (ng/mL)

Gefitinib M523595 M608236 M537194 M387783

1.00–200 1.00–200 0.50–100 1.00–200 1.00–200

y = 0.0327x + 0.0044 y = 0.0403x + 0.0029 y = 0.0709x + 0.0030 y = 0.0075x − 0.0004 y = 0.0123x − 0.0008

0.9997 0.9999 0.9999 0.9975 0.9999

1 1 0.5 1 1

LLOQ, lower limit of quantification.

Table 4 Precision and accuracy for the determining of gefitinib and its metabolites in mouse plasma (n = 3 days, five replicates per day). Compounds

Con. (ng/mL)

RSD (%)

RE (%)

Spiked

Measured

Intra-day

Gefitinib

3 25 160

3.14 ± 0.14 23.15 ± 1.85 158.69 ± 11.82

3.47 6.24 3.34

4.41 7.97 7.45

4.83 −7.40 −0.82

M523595

3 25 160

3.23 ± 0.35 24.40 ± 3.44 158.11 ± 16.49

4.87 6.64 5.90

10.83 14.10 10.43

7.58 −2.39 −1.18

1.54 ± 0.18 12.75 ± 0.98 76.81 ± 6.30

5.24 2.66 4.43

11.83 7.65 8.20

2.35 1.98 −3.99

1.5 12.5 80

M608236

Inter-day

M537194

3 25 160

3.07 ± 0.22 24.14 ± 2.85 161.21 ± 13.82

4.30 7.42 4.91

7.19 11.81 8.57

2.47 −3.43 0.76

M387783

3 25 160

3.11 ± 0.31 25.75 ± 3.44 157.89 ± 23.51

6.20 6.14 4.16

9.82 13.35 14.89

3.69 3.00 −1.32

RSD (%)

Extraction recovery (%)

Con., concentration. Table 5 Matrix effect and extraction recovery of gefitinib, its metabolites and IS in mouse plasma. Compounds

Spiked con. (ng/mL)

Matrix effect (%)

RSD (%)

Gefitinib

3.00 25.0 160

93.23 93.87 91.55

3.66 6.49 4.51

99.77 97.40 100.25

1.02 6.35 5.66

M523595

3.00 25.0 160

90.51 86.29 90.02

2.76 5.29 3.04

94.60 93.40 95.13

2.90 13.08 5.17

M608236

1.50 12.5 80.0

101.44 107.48 112.06

4.37 4.34 1.18

101.10 104.69 104.82

5.71 4.36 2.66

M537194

3.00 25.0 160

100.13 92.77 94.32

4.69 7.83 4.40

92.70 86.88 91.07

12.42 8.46 9.28

M387783

3.00 25.0 160

93.79 94.28 92.48

6.27 12.78 12.39

93.98 87.40 86.42

9.72 10.02 11.16

107.99

4.90

98.44

6.06

Dasatinib

20

Con., concentration.

provided by HFK bioscience Co., Ltd. (Beijing, China). All the mice were kept in environmentally controlled cages (23 ± 2 ◦ C; 12 h light/dark cycle and relative humidity 50%) at least 5 days prior to the experiments. The H1975 NSCLC cells were subcutaneously inoculated into the right flank of nude mice and let the tumor volume reaching approximately 50–100 mm3 until the experiment. Mice were allowed free access to standard laboratory diet and water, but fasted overnight before experiments. Mice were administrated with 50 mg/kg gefitinib which dissolved in tween 20, and the blood samples (0.5 mL) were collected from fosse orbital veins into heparinized polythene tubes at 0.25, 0.5, 1, 2, 3, 6, 8, 12, 16, 24 and 48 h, respectively. Each time point contains five mice. The supernatant

of blood samples was gathered after centrifuging at 4000 rpm for 10 min, and stored at −80 ◦ C until analysis. The PK analysis was performed by a non-compartmental approach using the WinNonlin version 6.3 (Pharsight Corporation, Mountain View, CA, USA).

3. Results and discussion 3.1. Mass method development In Fig. 1, we summarized the structure formula of gefitinib, its metabolites (M523595, M608236, M537194 and M387783) and IS corresponding mass spectra with their protonated molecular ion. In

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Table 6 Stability of gefitinib and its metabolites in mouse plasma (n = 6). Compounds

Spiked con. (ng/mL)

10 ◦ C for 24 h

RT for 6 h RE (%)

RSD (%)

RE (%)

Frozen for 30 days

Freeze–thaw

RSD (%)

RE (%)

RSD (%)

RE (%)

RSD (%)

Gefitinib

3 25 160

−1.18 −8.62 12.06

4.80 2.94 5.34

−7.59 1.4 9.22

4.78 3.16 3.37

7.47 −1.11 13.68

9.64 9.94 5.16

4.08 3.05 14.37

9.25 10.42 3.88

M523595

3 25 160

11.05 12.44 −0.45

4.45 2.87 7.33

3.49 −13.21 7.34

6.78 2.14 −0.53

10.48 6.13 14.92

1.83 10.20 3.87

−1.63 2.79 14.58

9.86 9.43 1.27

2.33 −2.44 6.44

4.50 2.32 2.92

12.12 −1.61 5.86

4.39 2.07 3.09

-0.65 10.17 −1.79

10.95 7.91 4.73

7.27 −8.27 2.23

8.49 7.52 4.01

M608236

1.5 12.5 80

M537194

3 25 160

8.17 7.05 14.82

10.86 2.67 5.93

8.23 9.03 −4.96

4.01 1.59 5.93

10.27 9.08 12.8

2.18 1.49 7.12

−5.34 7.78 12.49

5.28 11.37 6.23

M387783

3 25 160

−5.82 11.58 9.17

7.90 5.50 7.25

11.18 6.15 −9.13

4.67 2.82 7.60

2.67 1.99 13.85

5.40 3.81 2.21

−2.29 −14.94 −1.51

3.84 9.08 9.19

RT, room temperature; Con., concentration.

Table 7 Dilution integrity of gefitinib and its metabolites in mouse plasma (n = 6). Compounds

Gefitinib M523595 M608236 M537194 M387783

Dilution factor = 10

Dilution factor = 20

Con. (ng/mL)

RE (%)

RSD (%)

Con. (ng/mL)

RE (%)

RSD (%)

100 100 50 100 100

8.02 4.81 8.88 3.13 −4.14

5.7 4.98 0.97 6.78 2.75

50 50 25 50 50

2.07 1.50 8.51 1.82 0.60

3.12 2.99 1.32 2.09 3.55

Con., concentration. Table 8 Pharmacokinetic parameters for gefitinib and its metabolites in mice plasma after oral administration of 50 mg/kg gefitinib. Data are shown as mean ± SD (n = 5 for each time point). Parameters

Gefitinib

M387783

M523595

M537194

M608236

HL␭z (h) Tmax (h) Cmax (ng/ml) AUClast AUCinf AUC%extrap (%) AUMClast MRTlast (h)

4.92 ± 1.11 3.00 3127.13 ± 823.45 12485.44 ± 1097.12 12495.76 ± 1245.93 0.08 ± 0.02 64435.34 ± 2561.09 5.16 ± 1.98

– 3.00 4.64 ± 1.49 16.53 ± 4.82 – – 56.46 ± 19.45 3.42 ± 1.08

3.74 ± 0.92 3.00 365.98 ± 104.7 1588.21 ± 409.28 1603.07 ± 497.76 0.93 ± 0.28 9148.10 ± 1904.19 5.76 ± 1.85

6.07 ± 1.70 3.00 194.19 ± 78.24 655.43 ± 183.85 676.51 ± 200.18 3.12 ± 1.01 3558.54 ± 998.19 5.43 ± 2.01

6.46 ± 1.98 3.00 60.97 ± 20.53 197.80 ± 87.45 203.77 ± 79.24 2.93 ± 0.89 890.85 ± 194.28 4.50 ± 1.90

order to obtain better responses, the MS conditions were optimized with corresponding standard solutions. The effect of positive and negative ionization modes on the assay sensitivity of the analysts was investigated. The results showed that the ionization of gefibinib and its metabolites was more efficient in positive mode than the negative one. As shown in Table 2, the predominant protonated molecular ions [M+H]+ and the precursor → product ion transitions of gefitinib and its metabolites were listed, among which gefitinib and M523595 were consistent with the previous reports [11]. To obtain the maximum sensitivity for [M+H]+ ions detection, the fragment or together with suitable collision energy (CE) was optimized (Table 2), and 0.1% (v/v) formic acid was added into mobile phase helped to obtain favorite peak width and peak symmetry. 3.2. Chromatography Improved peaks resolution was accomplished using a linear gradient from 10% to 50% of acetonitrile in 2.0 min. The shortest analytical run time of the present method was 6 min although all compounds were eluted sufficiently within 3 min after injection.

The remaining 3 min was spent on diminishing a memory effect from the column and stabilizing it before next injection. Mobile phase with 95% eluent B for 1.5 min was introduced to diminish the memory effect and a re-equilibration phase of 10% B (v/v) had to be implemented to ensure the analytical column was stabilized. Typical chromatograms of blank plasma sample and samples at LLOQ are depicted in Fig. 2.

3.3. Specificity The specificity was fully assessed by screening the chromatograms of blank plasma of mouse, blank mouse plasma spiked with gefitinib and its metabolites as well as dasatinib (IS) (Fig. 2). At LLOQ level, a signal to noise ratio (S/N-ratio) of >5 was obtained for all analytes. No endogenous interference was observed at retention times of gefitinib (1.86 min), M523595 (1.82 min), M608236 (2.28 min), M537194 (1.80 min), and M387783 (1.46 min) or the IS (2.5 min). It indicated the developed method had efficient specificity under the working conditions.

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Fig. 3. The concentration–time profiles of gefitinib and its metabolites in mice plasma after oral administration of 50 mg/kg of gefitinib (n = 5 for each time point).

3.4. Linearity of calibration curves and LLOQs

3.8. Carry-over

The calibration curves and LLOQs of gefitinib and its metabolites are presented in Table 3. The developed method exhibited good linearity with excellent r ≥ 0.9998 for all analytes except M537194 was with r = 0.9987. The results indicated that the LLOQs were suitable for analytes determination in the PK studies.

We calculated the sample carry-over rate to ensure the precision for low concentration sample detection. The auto-sampler carry-over was minimized by using an acidic flush solvent (acetonitrile:methanol:isopropanol:water = 1:1:1:1, 0.1% formic acid) and increasing the rinse dip time from 5 to 15 s. The carry-over test fulfilled the criteria for dasatinib (0.1%), gefitinib (11.3%), M608236 (14.1%), M523595 (9.2%), M387783 (12.4%) and M537194 (14.5%).

3.5. Accuracy and precision As shown in Table 4, the intra-day and inter-day precisions (RSD) of QC samples at three different levels were both less than 15% and the accuracies (RE) ranged from −7.40% to 7.58% (Table 4). 3.6. Matrix effect and recovery As shown in Table 5, the matrix effect of the five analytes ranged from 91.55 to 93.87% for gefitinib, 86.29 to 90.51% for M523595, 101.44 to 112.06% for M608236, 92.77 to 100.13% for M537194 and 92.48 to 94.28% for M387783. The mean matrix effect of the IS was 107.99 ± 4.90%. The mean recoveries were between 86 and 105% for all analytes. These results indicated that there was no significant ion suppression in this method. Protein precipitation seemed to be a fast and simple one-step sample pretreatment procedure for the analytes in mouse plasma. 3.7. Stability The results of bench-top, auto-sampler, long-term and freeze–thaw studies revealed that all analytes achieved good stability in mouse plasma under different storage conditions expected during the routine analysis of the samples (Table 6). No significant decrease of signals was observed in the processed sample deviations at all QC levels were within 15% from the spiked concentration.

3.9. Dilution integrity Dilution integrity was examined by ten- and twenty-fold dilution of UHQC samples. The results had displayed that the accuracy and precision of the diluted samples were within the acceptance range (data are shown in Table 7). Therefore, the plasma samples exceeding the ULOQ could be adequately diluted with blank plasma by using the tested dilution factors before analysis. 3.10. Applicability of the assay for pharmacokinetic study After full validation, this method was proved to be simple and efficient in analyzing large batches of mouse plasma samples. In the PK study, we effectively detected all the five compounds in mouse administrated with 50 mg/kg gefitinib, which is translated from the dose used in the clinics [22]. The typical plasma concentration–time profile for gefitinib and its metabolites is presented in Fig. 3 and the corresponding PK parameters are listed in Table 8. The PK parameters for 5 compounds were calculated successfully, however, some of the PK parameters of M387783 were omitted due to the insufficient sensitivity of M387783 for the PK study. M387783 was a minor metabolite of gefitinib and eliminated rapidly. Due to the restriction of carry-over, the lower limit of quantification (LLOQ) was set at 1 ng/mL. As metabolism goes by, the concentration of M387783

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reached a peak at 3 h and was undetectable 12 h later after oral administration of gefitinib. The half-time (t1/2 ) of gefitinib was 4.92 h, which was in agreement with the literature reported t1/2 [16]. The peak time (Tmax ) of gefitinib was 3 h and it was similar to gefitinib in human pharmacokinetics trials [20,21]. However, the Tmax of four main metabolites of gefitinib was also 3 h. The area under concentration–time curve (AUC) of gefitinib was found to be 12,495.76 h ng/mL, while the AUC of metabolites followed by the order of M523595 > M537194 > M608236 > M387783. The peak concentration (Cmax ) of gefitinib was found to be 3127.13 ng/mL, while the metabolites followed by the same order as their AUC. The PK parameters indicated that gefitinib displayed good absorption and experienced rapid and extensive metabolism. 4. Conclusion A method has been developed and validated for simultaneous quantification of gefitinib and its four main metabolites in mouse plasma. The assay has been shown to have adequate specificity to estimate the PK profile of gefitinib and its metabolites in preclinical studies. This assay has been proved to be both robust and reliable. Moreover, simultaneous determination of gefitinib and its major metabolites in a single analytical run serves a high throughput of a heterogeneous batch of plasma samples of mouse on gefitinib metabolism study. Conflict of interest The authors declare that they have no conflict of interest in this study. Acknowledgements This research was supported by the National Natural Science Foundation of China, (No. 81274148) and Beijing Municipal Health System Special Funds of High-Level Medical Personnel Construction (No. 2014-3-063). References [1] M.H. Cohen, G.A. Williams, R.G. Sridhara, et al., United States Food and Drug Administration Drug Approval summary: gefitinib (ZD1839; Iressa) tablets, Clin. Cancer Res. 10 (2004) 1212–1218. [2] S. Dhillon, Gefitinib: a review of its use in adults with advanced non-small cell lung cancer, Target. Oncol. 10 (2015) 153–170. [3] E.S. Kim, V. Hirsh, T. Mok, et al., Gefitinib versus docetaxel in previously treated non-small cell lung cancer (INTEREST): a randomised phase III trial, Lancet 372 (2008) 1809–1818. [4] M. Maemondo, A. Inoue, K. Kobayashi, et al., Gefitinib or chemotherapy for non-small-cell lung cancer with mutated EGFR, N. Engl. J. Med. 362 (2010) 2380–2388. [5] D. McKillop, A.D. McCormick, A. Millar, et al., Cytochrome P450-dependent metabolism of gefitinib, Xenobiotica 35 (2005) 39–50.

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