Anal Bioanal Chem (2014) 406:1799–1805 DOI 10.1007/s00216-013-7570-1
Development and validation of a UPLC–MS/MS assay for the quantification of simotinib in human plasma Ning Li & Xiaohong Han & Ping Du & Yuanyuan Song & Xingsheng Hu & Sheng Yang & Yuankai Shi
Received: 12 July 2013 / Revised: 21 November 2013 / Accepted: 10 December 2013 / Published online: 10 January 2014 # Springer-Verlag Berlin Heidelberg 2014
Abstract Simotinib is a novel oral small-molecule tyrosine kinase inhibitor that has demonstrated equal or superior antineoplastic activities to erlotinib in preclinical studies. In support of a clinical pharmacokinetic study, a sensitive and accurate liquid chromatography (LC) method with mass spectrometry detection using multiple reaction monitoring (MRM) in positive ion mode was developed and validated for the quantification of simotinib in human plasma. The sample preparation procedure involved a simple protein precipitation with methanol. Erlotinib was used as the internal standard. The optimal chromatographic behavior was achieved on a Zorbax SB-C8 column (2.1 mm× 100 mm, 3.5 μm) using a mixture of 0.1 % formic acid with 10 mM ammonium formate/methanol (20:80, v/v) as the mobile phase. The total LC analysis time per injection was 4 min with a flow rate of 0.2 mL/min. The recovery was greater than 90 % and no significant matrix effect was observed. The assay was validated over the concentration range of 1–1,000 ng/mL. The intra- and interday precision and accuracy of the quality control samples at low, medium, and high concentration levels showed at most 9.4 % relative standard deviation (RSD) and −7.4 to 7.4 % relative errors (RE). Assay selectivity, freeze/ thaw stability, storage stability, and dilution effects were also assessed. The method is now used to support clinical pharmacokinetic studies in patients with non-small cell lung cancer (NSCLC) after oral administration of simotinib. Keywords Simotinib . UPLC–MS/MS . Human plasma . Validation N. Li : X. Han : P. Du : Y. Song : X. Hu : S. Yang : Y. Shi (*) Department of Medical Oncology, Cancer Institute/Hospital, Chinese Academy of Medical Sciences & Peking Union Medical College, Beijing Key Laboratory of Clinical Study on Anticancer Molecular Targeted Drugs, 17 Panjiayuan Nanli, Chaoyang District, Beijing 100021, China e-mail: [email protected]
Y. Shi e-mail: [email protected]
Introduction Lung cancer is the leading cause of cancer-related mortality worldwide, with nearly 1.4 million deaths each year . Nonsmall cell lung cancer (NSCLC) accounts for nearly 85 % of all cases of lung cancer , in which adenocarcinoma will be the most common histology. Chemotherapy for NSCLC remains marginally effective . Epidermal growth factor receptor (EGFR), a receptor tyrosine kinase (RTK) involved in cell differentiation, proliferation, angiogenesis, and apoptosis, is expressed in the majority of NSCLC. EGFR mutations are present in approximately 10 to 15 % of Caucasian patients and even higher percentage (up to 40 %) of Asian patients . Clinical trials have demonstrated that patients carrying activating mutations of EGFR significantly benefit from treatment with EGFR tyrosine kinase inhibitor (EGFR TKI), which is considered to be less toxic compared with the cytotoxic drugs. The prolonged progression-free survival (PFS) and increased disease control rates were observed when treated with EGFR TKIs in first-line compared to conventional platinum-based chemotherapy [5, 6]. The increased understanding of EGFR in promoting tumor angiogenesis has led to the rational development of EGFR TKIs. To date, three small-molecule EGFR TKIs, namely, erlotinib, gefitinib, and lapatinib, have gained US Food and Drug Administration (FDA) approval for use in oncology . Erlotinib and gefitinib were approved for the therapy of NSCLC, and lapatinib for the treatment of breast cancer . Icotinib hydrochloride (4-[(3-ethynylphenyl)amino]-6,7-benzo-12crown-4-quinazoline hydrochloride, BPI-2009H, Zhejiang Beta Pharma Inc., Hangzhou, China) , is a potent and reversible EGFR TKI that gained approval for treatment of NSCLC in China in 2011. Currently, several clinical trials related to icotinib have been initiated in China to evaluate its activity in NSCLC [10–13]. Simotinib, N-(3-chloro-4-fluorophenyl)-6-[2-(5, 8-dioxa10-azadispiro [18.104.22.168]undec-10-yl)ethoxy]-7-methoxy-4quinazolinamine (Fig. 1), is an innovative small-molecule
EGFR TKI (US 60/759,601, WO/2007/084875), which was chemically synthesized by Advenchen Laboratories LLC (Northridge, USA). Pharmacodynamic trial results indicated that simotinib has demonstrated equal or superior antineoplastic activities to erlotinib (unpublished results). Moreover, simotinib hydrochloride exhibited good tolerance among healthy subjects in a recent tolerance clinical trial with a single dose, which demonstrated that the highest safe dose was 500 mg (unpublished results). As a clinically promising compound, simotinib has been evaluated in an ongoing phase I clinical study in patients with NSCLC at the Cancer Institute/ Hospital, Chinese Academy of Medical Sciences since January 2013 (ClinicalTrials.gov identifier: NCT01772732). A few analytical methods using high-performance liquid chromatography with tandem mass spectrometry (LC–MS/ MS) have been developed and validated for determination of erlotinib  and icotinib , which have similar chemical structures to simotinib. In the aforementioned methods, liquid–liquid extraction (LLE) was used for pretreatment of samples for erlotinib  and icotinib , which was timeconsuming and could not satisfy the requirements of highthroughput determination in pharmacokinetic studies. Moreover, the matrix effect of icotinib using LLE was significant, and higher dilutions of the extraction residue were needed to eliminate the matrix effects. Recently, cost-effective HPLC– UV methods to quantify erlotinib in human plasma were reported; however, the lower limits of quantification (LLOQ) of the methods were 80 ng/mL  and 50 ng/mL , with single run times of 15 min  and 12 min , respectively. To the best of our knowledge, no analytical method for the determination of simotinib in biological samples using LC–MS/MS has been reported. In our study, we developed a rapid and sensitive method to quantify simotinib in human plasma with LC–MS/MS. A simple protein precipitation with methanol was used as the sample preparation procedure, and no significant matrix effect was observed. The LLOQ of the method is 1 ng/mL. The cycle time between two consecutive injections was approximately 4.0 min. This method was successfully applied to demonstrate the plasma concentration–time profile from one cancer patient receiving a twice daily oral administration of simotinib at 100 mg.
Experimental Chemicals and reagents Simotinib hydrochloride (C 25 H 26 ClFN 4 O 4 ·HCl, purity 99.8 %) and internal standard (IS) erlotinib hydrochloride (C22H23N3O4·HCl, purity 99.3 %) were supplied by Simcere Pharmaceutical Group (Jiangsu, China). Methanol (HPLC grade) was purchased from Fisher Scientific (Fair Lawn, NJ, USA). Formic acid (HPLC grade) and ammonium formate
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were purchased from Sigma-Aldrich (St. Louis, MO, USA). Ultra-pure water (0.22 μm) was deionized and further purified by means of a Milli-Q water purification system (Bedford, MA, USA) and used throughout the study. Heparinized blank (drug-free) human plasma was obtained from healthy volunteers. Instrumentation and analytical conditions Chromatography was performed on an Acquity UPLC system (Waters Corp., Milford, MA, USA) with an autosampler temperature set at 4 °C. Separation was performed at 25 °C on a Zorbax SB-C8 narrow bore RR column (2.1 mm×100 mm, 3.5 μm, Agilent). The mobile phase was 0.1 % formic acid with 10 mM ammonium formate/methanol (20:80, v/v) at a flow rate of 0.2 mL/min. A 2.0-μL aliquot was injected into the UPLC– MS/MS system, and the total analytical run time was 4 min. Mass spectrometric detection was performed on an API 5500QTrap (Applied Biosystem/MDS SCIEX, Foster City, CA, USA) equipped with a TurboIonSpray electrospray ionization (ESI) source and a positive-ion, multiple reaction monitoring (MRM) mode. The settings of the mass spectrometer were as follows: ionspray voltage, 5,500 V; ionspray temperature, 450 °C; curtain gas (CUR), nitrogen, 15; ion source gas 1 (GS1), 25; ion source gas 2 (GS2), 30; declustering potential (DP), 135 V; entrance potential (EP), 7 V; collision cell exit potential (CXP), 13 V. The analytes were detected by monitoring the transitions m/z 501.2→182.2 for simotinib and m/z 394.1→ 278.2 for erlotinib with a collision energy (CE) of 35 V. Product scan mass spectra of the analytes are shown in Fig. 1. All the parameters of LC and MS were controlled by Analyst 1.5.1 software (Applied Biosystem/MDS SCIEX). Standards Two stock solutions of simotinib were prepared separately in methanol at a target concentration of 1 mg/mL. One solution was used for calibration standards and the other was used for quality control (QC) samples. Serial dilution with methanol was performed to achieve the desired concentration of working solutions starting from the stock solutions. Similarly, an IS stock solution (1 mg/mL) was also prepared in methanol and diluted with methanol to give an IS working solution (50 ng/ mL). All stock solutions were stored at −20 °C until utilized. A 20-μL aliquot of IS working solution and 5 μL simotinib working solution were successively added into 1.5-mL prelabeled microcentrifuge tubes each containing 50 μL drugfree human plasma. Samples were vortex-mixed for approximately 10 s. The plasma concentrations of simotinib on the calibration curve were 1, 3, 10, 30, 100, 300, and 1,000 ng/mL. Final concentrations at LLOQ, low QC, medium QC, high QC, and QC samples above the upper limit of quantification (>ULOQ) were 1, 2, 40, 800, and 2,000 ng/mL, respectively.
Development and validation of a UPLC–MS/MS assay for the quantification of simotinib in human plasma
Fig. 1 Chemical structures and mass spectra of simotinib (left) and erlotinib (internal standard, right)
Blank sample (IS only) was spiked with 20 μL IS working solution and 5 μL methanol into 50 μL drug-free plasma. Double blank (no simotinib and IS) sample was produced by adding only 25 μL of methanol to 50 μL drug-free plasma. Sample preparation Clinical plasma samples were thawed, allowed to equilibrate at room temperature, and thoroughly vortexed prior to analysis. A 20-μL aliquot of IS working solution (50 ng/mL) and 5 μL of methanol were successively added to 50 μL of clinical plasma sample. After the samples were vortex-mixed for 10 s, 400 μL of methanol was added to the blank, calibration standards, QCs, and clinical plasma samples, respectively. The mixture was vortexed to precipitate the proteins. Then, the samples were centrifuged at 12,000 rpm for 10 min at 4 °C. Clear supernatants were transferred to glass autosampler vials and an aliquot of 2 μL was injected into the LC–MS/MS system for analysis. Method validation A complete validation was performed for the assay in biological samples according to FDA guidance . Validation involved evaluation of specificity, linearity, precision and accuracy, lower limit of quantification, carry-over effect, recovery, matrix effects (ion suppression/enhancement), stability, and dilution integrity.
Results and discussion LC–MS/MS method development and sample preparation Positive ionization with ESI was better than negative ionization for LC–MS/MS detection of simotinib. ESI parameters such as ionspray temperature, ionspray voltage, CUR, GS1 and GS2 of simotinib and IS were optimized to obtain maximum precursor ion intensity. The CE and CXP were both optimized to obtain the maximum daughter ion intensity. Figure 1 shows the final transitions that were selected and their proposed fragmentation patterns for simotinib and IS.
In order to get a good separation and peak shape we tested several C18 reversed-phase columns with a broad pH range, namely, Zorbax Eclipse Plus C18 (50 mm×4.6 mm and 50 mm×2.1 mm , 3.5 μm, Agilent), Acquity UPLC BEH C18 (50 mm×2.1 mm, 1.7 μm, Waters), and Acquity BEH Shield RP 18 (100 mm×2.1 mm, 1.7 μm, Waters), but severe peak-tailing and carry-over (an area of 120–160 % of the LLOQ in the first blank plasma after ULOQ sample and an area of 80–100 % of the LLOQ in the second blank plasma after ULOQ sample) was observed when these conventional C18 reversed-phase columns were applied. Since simotinib is a weakly basic compound with little polarity, the optimum peak shape and minimized carry-over of simotinib were achieved on a Zorbax SB-C8 column (100 mm×2.1 mm, 3.5 μm, Agilent) using isocratic elution. The separation was optimized by adjusting the ratio of methanol to the formate buffer with 10 mM ammonium formate in the mobile phase. A good peak shape and separation were both obtained with a retention time at 1.8 min for simotinib and 1.9 min for IS when the mobile phase was 80 % methanol/formate buffer containing 0.1 % formic acid. A switching valve was used to divert the flow to the waste container for the first and last minute, and only the flow from the column from 1.0 to 3.0 min was introduced into the mass spectrometer. To minimize the carry-over, the autosampler was washed with strong needle washes (600 μL methanol) and weak needle washes (1,800 μL mobile phase) before and after each injection, the volume of which were both tripled compared with the general method. During the development of the sample preparation procedure, protein precipitation (PPT) with methanol and acetonitrile were both tested. The extraction recovery by the use of methanol was found to be higher and more reproducible than the use of acetonitrile. The LLE with tert-butyl methyl ether (TBME) was also investigated with equal recovery to protein precipitation with methanol. Therefore, a simple protein precipitation step with methanol was preferred in the study because it is simple, rapid, and most cost-effective. Erlotinib was selected as IS due to its similar ionization condition, extraction efficiency, and chromatographic and MS behaviors compared to simotinib. Erlotinib appeared to be stable under the described experimental conditions and did not interfere with the analysis of simotinib.
Validation results The linearity range was first tried from 1 to 3,000 ng/mL. However, the increase of simotinib concentration was higher than that of the mass spectra response. Hence, we set the ULOQ at 1,000 ng/mL and an acceptable linearity was Fig. 2 Typical MRM chromatograms for selected extracted samples (left simotinib, right IS, erlotinib): double-blank, blank sample, LLOQ, and response for patient’s sample (conc. 8.87 ng/mL; 0.5 h after oral administration on day 1)
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achieved in the range of 1–1,000 ng/mL for simotinib. Seven non-zero calibration standards were prepared and analyzed in three analytical runs. The r2 value was greater than 0.995 in all validation batches. The S/N ratios of the LLOQ samples were all greater than 15. Simotinib and IS was eluted at 1.88 min and 1.91 min, respectively, and no significant interfering peaks
Development and validation of a UPLC–MS/MS assay for the quantification of simotinib in human plasma Table 1 Precision (RSD) and accuracy (RE) values for quantification of simotinib in plasma (n=6)
Values are expressed as percentages and indicate the variation and the deviation from the real concentration (100 %), respectively
1.00 2.00 40.0 800
Day 1 (n=6)
Day 2 (n=6)
Day 3 (n=6)
Interday (day=3, n=18)
9.4 3.1 4.9 1.1
−7.4 7.4 5.7 1.9
5.4 2.8 3.3 2.4
−6.8 −5.0 −2.8 −3.1
6.3 3.7 1.2 2.8
−5.6 −7.2 −1.8 −7.4
6.5 7.4 5.1 4.5
−6.8 −1.6 0.4 −2.9
were observed at these retention times. Representative chromatograms of blank plasma and plasma spiked at the LLOQ are presented in Fig. 2. The precision and accuracy of the method were determined by analyzing the QC samples at low, medium, and high concentrations. As shown in Table 1, good precision and accuracy could be achieved for this assay under the current method validation conditions. For determining the matrix effects, the analytical response of blank plasma extracts from six different sources spiked with simotinib (presence of matrix ions) was compared with the response of a neat solution (absence of matrix ions) spiked with simotinib. The matrix effects determined at concentrations of 2, 40, and 800 ng/mL for simotinib were 96.0 % (CV %, 6.9), 103.0 % (CV %, 5.0), and 92.3 % (CV %, 7.2), respectively, showing no concentration dependency. The matrix effect for IS at a concentration 15 ng/mL was 95.7 % (CV %, 3.4), which was evaluated in the same way. Overall, the results showed that co-eluting matrix components did not affect the ionization of simotinib and IS. The extraction recovery was calculated as the ratio of the analytical response obtained from analysis of extracted spiked matrix samples (prespike) relative to the analytical response obtained from analysis of the extracted blank samples spiked after extraction with reference solutions (postspike). The extraction recoveries of the same three QC samples and IS were 91.2 % (CV %, 2.2), 87.2 % (CV %, 8.1), 91.3 % (CV %, 5.0), and 87.4 % (CV %, 1.3), respectively. Therefore, the extraction recovery of simotinib and IS was consistent, precise, and reproducible.
Table 2 Stability of simotinib (n=5)
Carry-over effect was investigated by injecting three processed double blank plasma samples subsequently after injection of a ULOQ sample in three independent runs. The responses were evaluated at the retention time of simotinib and IS. An area of less than 5 % of the LLOQs in plasma was observed in the first blank plasma sample after a ULOQ sample at the retention time of simotinib, and no response peaks at the same retention time of IS was observed with this assay. Therefore, the carry-over effect was found to be negligible from previous concentrated samples. The analyte stability tests were designed to cover the expected handling conditions of clinical samples under a variety of storage and processing conditions. The QC samples stored at −80 °C remained stable for at least 5 months in plasma. No significant degradation of simotinib was found in plasma after three freeze/thaw cycles. The stability of simotinib in the final extract at 4 °C and 22 °C was monitored for at least 48 h and 17 h, respectively. The concentration deviations were within 15 % for all levels of QCs (Table 2). The percentage recoveries of stock solutions of simotinib and IS stored at −20 °C for 70 days compared with a freshly prepared stock solution agree within 4.8 %, showing the stock solutions of simotinib and IS were both stable. A human plasma QC sample at 2,000 ng/mL was diluted tenfold using blank human plasma and analyzed for simotinib. The accuracy of mean back-calculated concentrations for tenfold dilution of simotinib was less than ±4.2 % of their nominal concentration. The precision for tenfold dilution samples was less than 13.1 %.
Stability test conditions
Nominal concentration (ng/mL) 2.00
Values expressed as mean (% deviation)
Short-term stability of plasma (22 °C, 17 h)
Long-term stability of plasma (−80 °C, 5 months) Short-term stability of plasma extract (22 °C, 17 h) Short-term stability of plasma extract (autosampler 4 °C, 48 h) Three freeze/thaw cycles
2.22 (11.0) 2.27 (13.6) 1.86 (−7.0) 2.29 (14.5)
42.15 (5.4) 39.61 (−1.0) 41.3 (3.2) 45.0 (12.6)
778.83 (−2.6) 793.16 (−0.9) 770.8 (−3.6) 817.8 (2.2)
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Fig. 3 Concentration versus time curves of simotinib in plasma from a patient after a single dose of 100 mg on day 1, predose on days 8, 9, and 10, and multiple dose (100 mg twice daily) on day 15
A UPLC–MS/MS method for the determination of simotinib in human plasma was developed and validated with respect to sensitivity, accuracy, precision, and reproducibility. The use of a Zorbax SB-C8 column (2.1 mm×100 mm, 3.5 μm) significantly improved the retention and peak shape of simotinib. The validated linear range for simotinib was 1–1,000 ng/mL with an LLOQ of 1 ng/mL, using 50 μL human plasma aliquots. The simplicity of sample preparation using protein precipitation and a short chromatographic run time (4 min) make the method suitable for high-throughput determination in clinical pharmacokinetic analysis. This method was proven to be reproducible, robust, and ready to be applied to the clinical pharmacologic studies of simotinib in patients with NSCLC.
Clinical application The purpose of the validated assay was to support the clinical pharmacokinetic investigations of simotinib in cancer patients receiving a twice daily oral administration. The pharmacokinetic study was submitted to the Cancer Hospital Ethical Committee and subjects provided written informed consent before entering this study. Subject confidentiality was maintained throughout the trial. Patients were excluded if they had been treated by any EGFR TKIs such as erlotinib, gefitinib, sunitinib, and sorafenib. In addition, previous anticancer chemotherapy had to be discontinued for at least 4 weeks before entry into the study. To demonstrate the applicability of the proposed assay, we present the concentration profiles over time of simotinib in plasma of a representative patient who had received an oral dosage of 100 mg simotinib twice daily until disease progression or undue toxicity was observed. The starting dosage of 100 mg twice daily corresponds to the data obtained in healthy human volunteers. Blood samples for pharmacokinetic measurements were taken on day 1 after the first dose (equivalent to a single-dose evaluation) and on day 15 for multiple-dosing evaluation; measurement times included the following: predose and at 0.5, 1, 1.5, 2, 4, 6, 8, 10, and 12 h on days 1 and 15. On days 8, 9, and 10, predose samples were collected to check the trough level. All blood samples were immediately collected in heparinized tubes. Plasma was separated by centrifugation of the whole blood sample at 3,000 rpm for 10 min and stored at −80 °C until subsequent analysis. The plasma concentration–time curve of simotinib in a patient after single and multiple dosing of 100 mg simotinib is presented in Fig. 3. The results indicated that this method was sufficiently sensitive for analyzing simotinib in human plasma for up to 12 h after the administration of a single dose of 100 mg. The concentrations of simotinib on days 8, 9, and 10 predose samples were 38.2, 40.3, and 42.6 ng/mL, respectively, which was consistent with the last detectable plasma concentration (Clast) 31.8 ng/mL on day 15 indicating a probable steady state.
Acknowledgments This study was sponsored by Jiangsu Simcere Pharmaceutical R&D Co. Ltd (Jiangsu, China). It was partly funded by the Chinese National Science and Technology Major Project for New Drug Innovation (2012ZX09303012, 2012ZX09105-301002 and Beijing Municipal Science and Technology Commission Major Project for New Drug Innovation (Z111102071011001, Z121102009212055). Conflict of interest No potential conflict of interest was disclosed.
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