Simultaneous Quantification of Brigatinib and Brigatinib-Analog in Rat Plasma and Brain Homogenate by LC-MS/MS: Application to Comparative Pharmacokinetic and Brain Distribution Studies

Brigatinib and brigatinib-analog are potent and selective ALK inhibitors with the similar structure. A simple and sensitive high-performance liquid chromatography with tandem mass spectrometry (LC-MS/MS) method for simultaneous determination of brigatinib and brigatinib-analog in rat plasma and brain homogenate was developed and validated. Chromatographic separation was carried out on an ODS column with acetonitrile and 0.1% formic acid in water as the mobile phase with gradient elution at a flow rate of 0.5 mL/min. Detections were performed using a TSQ Quantum Ultra mass spectrometric detector with electrospray ionization (ESI) interface, which was operated in the positive ion mode. A simple protein precipitation preparation process was used. The lower limits of quantification (LLOQs) were 1.0 ng/mL and 0.5 ng/mL for analytes in rat plasma and brain homogenate, respectively. The intrabatch and interbatch precision and accuracy of brigatinib and brigatinib-analog were well within the acceptable limits of variation. The simple and sensitive LC-MS/MS method was successfully applied to the pharmacokinetic and brain distribution studies following a single oral administration of brigatinib and brigatinib-analog to rats. The above studies would lay a good foundation for the further applications of brigatinib and brigatinib-analog.


Introduction
Lung cancer is one of the most commonly diagnosed tumors with high morbidity and mortality worldwide [1], and its incidence and mortality continue to grow. Lung cancer is divided into small-cell lung cancer (SCLC) and non-smallcell lung cancer (NSCLC), of which NSCLC accounts for 85% of lung cancer, including squamous cell carcinoma, adenocarcinoma, and large cell undifferentiated cancer [2,3]. SCLC accounts for only 15%. Compared with SCLC, NSCLC has slower metastasis and proliferation. About 75% of NSCLC patients were found to be at the middle and late stages, and the 5-year reported survival rate was extremely low [4,5].
e development of molecular research in the nearly ten years has meant significant breakthroughs in the diagnosis, detection, and treatment of lung cancer of the NSCLC. Anaplastic lymphoma kinase (ALK), a receptor tyrosine kinase of the insulin receptor family, was originally identified as a part of the fusion protein nucleophosmin-anaplastic lymphoma kinase (NPM-ALK). e gene rearrangement between ALK and echinoderm microtubule-associated protein-like 4 (EML4) becomes more general than ALK gene rearrangement, which is no more than 5% in advanced NSCLC. e constitutive kinase activity of the final product with carcinogenicity (EML4-ALK) represents the growth of ALK-rearranged (ALK-positive) NSCLC [6][7][8].
As one of the second-generation ALK inhibitors, brigatinib (AP26113), approved by FDA in April 2017, is a highly selective and efficient ALK inhibitor to treat patients with ALK-positive metastatic NSCLC and can overcome the acquired crizotinib resistance to the first-generation ALK inhibitor, especially the L1196M gatekeeper mutation [9,10]. Brigatinib-analog (AP26113-analog, ALK-IN-1) is an orally active, potent and selective ALK, and the epidermal growth factor receptor (EGFR) inhibitor with the similar structure to brigatinib. Brigatinib-analog binds to and inhibits ALK kinase and ALK fusion proteins as well as EGFR and mutant forms. is leads to the inhibition of ALK kinase and EGFR kinase, disrupts their signaling pathways, and eventually inhibits tumor cell growth in susceptible tumor cells [3,11].
It was reported that brigatinib and brigatinib-analog had similar potency against the triple mutation with IC50 values of <100 nM. In addition, the brigatinib and brigatinib-analog play a therapeutic role for brain metastases due to ability to reach the central nervous system (CNS) through the blood-brain barrier [12,13].
To the best of our knowledge, there were only a few studies reported to determine the concentration of brigatinib in plasma and tissues [14,15], and this was the first research reported for simultaneous determination of brigatinib and brigatinib-analog in rat plasma and brain homogenate using an LC-MS/MS method. As the primary purpose of the study, a sensitive, reliable, and simple LC-MS/ MS method was established and validated for the simultaneous quantification of brigatinib and brigatinib-analog in rat plasma and brain homogenate. Also, the method was successfully applied to the pharmacokinetic and brain distribution studies of brigatinib and brigatinib-analog following a single oral administration to rats.
is research would provide the foundations for further applications of brigatinib and brigatinib-analog.

Instruments and LC-MS/MS Conditions.
Analyses were acquired on a TSQ Quantum Ultra mass spectrometric detector with electrospray ionization (ESI) interface ( ermo Scientific, USA), which was coupled with a Dionex Ultimate 3000 ultra-performance liquid chromatography system consisting of an LPG-3400SDN pump, a WPS-3000TSL autosampler, and a TCC-3000RS column compartment. Samples were separated on a reversed phase Inertsil ODS-3 column (50 mm × 4.6 mm ID, 5 μm particle size, GL Sciences Inc.) at 25°C and eluted on a gradient mobile phase of water (containing 0.1% formic acid) and acetonitrile at a flow rate of 0.5 mL/min. e consecutive program was as follows: 15% acetonitrile followed by a linear increase to 20% acetonitrile over a 0.2 min period, followed by a linear gradient elution of 20% to 50% acetonitrile from 0.2 to 1.9 min, finally returned to 15% acetonitrile during 0.01 min, and re-equilibrated to its starting conditions until 3 min. e retention times were 2.07, 2.18, and 2.40 min for brigatinib, brigatinib-analog, and the IS, respectively. e acquisition was performed using selected reaction monitoring ( Figure 1). All the data were acquired and processed using Xcalibur software ( ermo Scientific, USA).

Preparation of Standard Solutions, Calibration Samples,
and Quality Control Samples. Primary stock standard solutions of brigatinib and brigatinib-analog were prepared separately for use as standard and quality controls (QC) at the concentration of 10.0 mg/mL with ethanol and mixed in equal volumes. e mixed standard solution was further serially diluted with methanol. e IS stock standard solution was prepared with dimethyl sulfoxide (DMSO) at the concentration of 1.0 mg/mL and further diluted with methanol to 1000 ng/mL. All standard solutions were kept at 4°C before use.
Calibration standards containing brigatinib and brigatinib-analog were prepared by spiking appropriate amounts of the standard solutions in rat blank plasma or brain homogenate (1 : 99, v/v). Seven levels of the calibration curve were determined (n � 5). e calibration equations were calculated by the leastsquares linear regression method. e final concentrations in plasma were 1.0, 4.0, 20, 80, 500, 1000, and 2000 ng/mL for both brigatinib and brigatinib-analog. Quality control (QC) samples were similarly prepared with blank plasma and the final concentrations were 1.0, 2.0, 400, and 1600 ng/mL. e final concentrations in the brain homogenate were 0.5, 2.0, 10, 40, 250, 500, and 1000 ng/mL for both brigatinib and brigatinibanalog. QC samples were both at the concentrations of 0.5, 1.0,    International Journal of Analytical Chemistry 200, and 800 ng/mL. All the spiked samples were treated in accordance with the biosample preparation procedure.

Biosample Preparation.
30 μL of rat biosample (plasma or brain homogenate) was mixed with 100 μL of IS solution and 150 μL methanol for protein precipitation. en, the mixture was vortex-mixed for 1 min and centrifuged at 14,000 rpm for 10 min at 4°C. After centrifugation, the supernatant was carefully separated, and a 10 μL aliquot was injected into the LC-MS/MS system for analysis.

Method Validation.
Validation procedures of the developed method were carried out according to US Food and Drug Administration (FDA) guidelines for bioanalytical method validation with respect to selectivity, sensitivity, linearity, intrabatch and interbatch precision and accuracy, recovery, matrix effect, stability, and carryover [16]. For the pharmacokinetic studies, after fasting with free access to water for at least 12 h, twelve rats were randomly divided into two groups (n � 6). e brigatinib and brigatinib-analog were both dissolved in ethanol-0.5% methylcellulose (1 : 19) for dosing. e two group rats were intragastrically administered with brigatinib and brigatinibanalog at the same dose of 5 mg/kg, respectively.

Application to Pharmacokinetic and Brain Distribution
Blood samples, about 100 μL of each, were placed in heparinized Eppendorf tubes predose and at 5, 15, 30, 60, 120, 180, 240, 300, 360, 480, 720, and 1,440 min postdose from the jugular vein of rats. e blood samples were immediately centrifuged at 3,500 rpm for 15 min at 4°C, and the plasma samples were transferred carefully from each collection and stored at − 20°C until analysis [17,18].
For the brain distribution studies, the remaining sixty rats were randomly divided into ten groups (n � 6, per group). e distributions of brigatinib and brigatinib-analog in the rat brain were also investigated at the dose of 5 mg/kg after single oral administration to rats. e whole brain was excised immediately at 5, 60, 240, 480, and 1,440 min postdose, thoroughly rinsed with 0.9% saline solution, and wiped with a filter paper. Each brain sample was weighed accurately and homogenized in double distilled water (1 : 5, w/v) by using a tissue homogenizer (IKA, Germany) and frozen at − 20°C until analysis [19].   International Journal of Analytical Chemistry 5 Differences between two groups were evaluated by the t-test. A value of p < 0.05 was considered as statistically significant.

Optimization of LC-MS Conditions
3.1.1. Mass Spectra. e analytes and IS were found to respond best to positive ionization with the adduct ions [M + H] + , which were presented as the major peaks. eir product ion mass spectra are shown in Figure 1. e SRM mode was used to monitor the transitions of m/z 584.26 ⟶ 484.08 for brigatinib, m/z 529.05 ⟶ 483.92 for brigatinib-analog, and m/z 500.24 ⟶ 71.89 for IS.

Liquid Chromatography.
When selecting the mobile phase for the LC-MS/MS system, attention was paid to the influence of the mobile phase on the chromatographic retention and the MS response. Formic acid modifier can improve the ionization efficiency. Different concentrations of formic acid at levels of 0.05%, 0.1%, and 0.2% were examined in the aqueous portion of the mobile phase. e results indicated that adding 0.1% formic acid in the aqueous portion was sufficient to achieve the highest MS sensitivity for brigatinib, brigatinib-analog, and IS. After lots of preliminary experiments had been done, it was found that an Inertsil ODS-3 column (50 mm × 4.6 mm ID, 5 μm particle size) produced the best separation of the two compounds. e mobile phase consisted of water (containing 0.1% formic acid) and acetonitrile with gradient elution, which was used for narrowing the peaks and improving the resolution of analytes and IS and shorting the running time of the chromatography.
In addition, owing to the complex nature of rat plasma and brain homogenate, the pretreatment of biosample is a significant step to remove protein and potential interferences prior to LC-MS/MS analysis. e extraction conditions, including protein precipitation, liquid-liquid extraction, and solid-phase extraction were investigated. e specificity, sensitivity, stability, and accuracy of protein precipitation, which required less time and decreased the cost of the assay, could meet the requirements of the research.

Selectivity.
Six different batches of blank biosample obtained from six different sources were prepared. No interferences from endogenous substances of plasma and brain homogenate were observed in the retention regions of either analytes or IS (Figure 2 and Supplementary materials Figure 1).

Linearity and LLOQ.
e calibration curves were obtained by plotting the peak area ratio of the analytes to IS against the corresponding concentration of the analytes in the freshly prepared rat biomatrix calibrators. e linear regression equations for brigatinib and brigatinib-analog in rat plasma were y � 5.65 × 10 − 4 x + 2.81 × 10 − 4 and y � 5.21 × 10 − 4 x + 5.78 × 10 − 4 , respectively. e mean correlation coefficients (r 2 ) of calibration curves were all >0.99 over the concentration range from 1.0 to 2000 ng/mL with the weighting factor of 1/x 2 . e linear regression equations for brigatinib and brigatinib-analog in the rat brain homogenate over the concentration range from 0.5 to 1000 ng/mL with the weighting factor of 1/x 2 were y � 7.43 × 10 − 4 x − 2.23 × 10 − 4 (r 2 � 0.9904) and y � 6.51 × 10 − 4 x + 2.33 × 10 − 4 (r 2 � 0.9919), respectively. e lower limits of quantification (LLOQs) for brigatinib and brigatinib-analog were both found to be 1.0 ng/mL in rat plasma and they were 0.5 ng/mL in the rat brain homogenate.

Precision and Accuracy.
e four different QC levels were used to assess the intrabatch and interbatch precision and accuracy of brigatinib and brigatinib-analog in rat plasma and brain homogenate by analyzing six replicates of each QC level on three separate batches using independently prepared calibration curves. e assay values were less than 15% for all concentrations (<20% for LLOQ) and conformed to the accepted variable limits, which demonstrated that the method was reliable and reproducible for quantification of brigatinib and brigatinib-analog in rat plasma and brain homogenate (Table 1).

Extraction Recovery and Matrix Effect.
e responses of analytes from low, medium, and high QC samples with known amounts (A), analytes dissolved in the postextracted blank matrix at the same QC concentration levels (B), and neat standards at the same QC concentration levels (C) were measured, respectively (n � 3). e ratio of A against C was used to estimate the extraction recovery, while the ratio of B against C was used to evaluate the matrix effect (Table 2). e results suggested that the extraction recovery was within the acceptable range, and there was no significant matrix effect on brigatinib and brigatinib-analog in this method.

Stability and Carryover.
e stability of brigatinib and brigatinib-analog was evaluated by analysis of low and high concentration levels of QC samples under different conditions (n � 6). e temperature and timing conditions were as follows: 6 h at ambient temperature, stored at − 20°C for 10 d, three freeze-thaw cycles, and 24 h at 4°C in an autosampler. e measured concentrations were all within acceptable limits (±15% of the nominal concentrations) during the entire validation. e results indicated that brigatinib and brigatinib-analog were stable under conditions investigated in this study (Table 3). Carryover was evaluated by determining the peak areas for brigatinib and brigatinib-analog by injecting blank matrix samples sequentially after the highest calibration standard sample injection. Furthermore, no carryover effect was obtained in this study.

Pharmacokinetic and Brain Distribution Application.
e validated method was successfully applied to study the pharmacokinetics of brigatinib and brigatinib-analog in rats by gavage at the dose of 5 mg/kg, respectively. 734.41 ± 83.06 ng/mL), apparent volume of distribution (V d ; 5.66 ± 1.94 vs. 3.43 ± 0.96 L/kg), and clearance (CL z /F; 0.022 ± 0.010 vs. 0.021 ± 0.006 L/min/kg) were observed between brigatinib and brigatinib-analog in rats. e mean Table 1: e intrabatch and interbatch precision and accuracy of brigatinib and brigatinib-analog in rat plasma and brain homogenate (n � 3 batches, 6 replicates per batch).    Figure 3. e established LC-MS/MS method was also successfully applied to investigate the brain distribution of brigatinib and brigatinib-analog in rats following oral administration of 5 mg/kg. e brain distribution researches found that the brigatinib and brigatinib-analog could both penetrate the blood-brain barrier (BBB). e ability of analytes penetrating the BBB was estimated by using the ratio of average AUC (0-t) in the rat brain to that in the plasma (AUC brain /AUC plasma ) at the same dose, supposing 1 g of brain tissue was equivalent to 1 mL of plasma. e ratio of AUC brain /AUC plasma was 0.223 and 0.088 for brigatinib and brigatinib-analog, respectively, which meant that brigatinib has stronger ability passing through the BBB than brigatinib-analog in rats. In addition, there were significant differences in AUC (0-t) , MRT, C max , V d , and CL z /F between brigatinib and brigatinib-analog in the rat brain (p < 0.05). e mean brain tissue concentrationtime profiles of brigatinib and brigatinib-analog following single oral administration in rats are presented in Figure 4. e main pharmacokinetic parameters of brigatinib and brigatinib-analog in rat plasma and brain tissue are presented in Table 4.

Conclusion
In conclusion, a highly sensitive, reliable, and simple LC-MS/MS method had been developed and validated for the simultaneous determination of brigatinib and brigatinibanalog in rat plasma and brain homogenate. e validated LC-MS/MS method was also successfully applied to the pharmacokinetics and brain distribution studies of brigatinib and brigatinib-analog in rats after a single oral administration of 5 mg/kg. e pharmacokinetic researches suggested that there remained some differences in pharmacokinetic characteristics between brigatinib and brigatinib-analog in rats. Moreover, compared with brigatinibanalog, brigatinib was a stronger central nervous systempenetrant. However, the future study should increase sample size to make the empirical conclusions more convincing. e above studies would lay a good foundation for the further applications of brigatinib and brigatinib-analog.
Data Availability e datasets generated and analysed during the current study are not publicly available due to confidentiality agreement of the institution but are available from the corresponding author on reasonable request.

Conflicts of Interest
e authors have declared no conflicts of interest.

Authors' Contributions
Bo Li and Min Lu contributed equally to this work.

Supplementary Materials
Supplementary Figure 1: SRM chromatograms for brigatinib (I) and brigatinib-analog (II) and IS in the rat brain homogenate: (A) blank rat brain homogenate; (B) blank brain homogenate spiked with the analytes (0.5 ng/mL) and IS; (C) a rat brain homogenate sample collected 4 h after single oral administration of 5.0 mg/kg brigatinib; (D) a rat brain homogenate sample collected 4 h after single oral administration of 5.0 mg/kg brigatinib-analog. (Supplementary Materials)