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.
Lung cancer is one of the most commonly diagnosed tumors with high morbidity and mortality worldwide [
The 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). The 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. The constitutive kinase activity of the final product with carcinogenicity (EML4-ALK) represents the growth of ALK-rearranged (ALK-positive) NSCLC [
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 [
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 [
To the best of our knowledge, there were only a few studies reported to determine the concentration of brigatinib in plasma and tissues [
Brigatinib (AP26113, CAS: 1197953-54-0, 99% of purity), brigatinib-analog (AP26113-analog, ALK-IN-1, CAS: 1197958-12-5, 99% of purity), and osimertinib (IS, CAS: 1421373-65-0, 98% of purity) were kindly supplied by the Institute of Materia Medica, Shandong First Medical University & Shandong Academy of Medical Sciences (Jinan, China). HPLC-grade methanol was obtained from Tedia (Fairfield, OH, USA), and acetonitrile, of HPLC grade, was supplied by Fisher Scientific (Fair Lawn, NJ, USA). HPLC-grade formic acid was purchased from Damao Chemical Reagent Factory (Tianjin, China). All other chemicals were of analytic grade or better.
Analyses were acquired on a TSQ Quantum Ultra mass spectrometric detector with electrospray ionization (ESI) interface (Thermo 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
The acquisition was performed using selected reaction monitoring (SRM) of the transitions from protonated precursor ion [M + H]+ to the particular daughter ion to quantify each compound. The SRM conditions were defined as follows: spray voltage 3500 V, vaporizer temperature 250°C, sheath gas pressure 35 arb, auxiliary gas pressure 10 arb, and capillary temperature 350°C. The SRM mode of
Chemical structures and product ion scan spectra for (a) brigatinib (I); (b) brigatinib-analog (II); (c) IS.
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. The mixed standard solution was further serially diluted with methanol. The 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 (
The calibration equations were calculated by the least-squares linear regression method. The 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. The final concentrations in the brain homogenate were 0.5, 2.0, 10, 40, 250, 500, and 1000 ng/mL for both brigatinib and brigatinib-analog. QC samples were both at the concentrations of 0.5, 1.0, 200, and 800 ng/mL. All the spiked samples were treated in accordance with the biosample preparation procedure.
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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 [
Seventy-two male Sprague Dawley rats weighing 180–220 g (7-8 weeks old, Certificate no. SCXK (Shandong) 2014-0007) were supplied by Shandong Laboratory Animal Center, Shandong First Medical University & Shandong Academy of Medical Sciences. All rats were kept in plastic cages with 12 h day/night cycle under controlled temperature (about 23–25°C) and fed with standard laboratory rat diet and water
For the pharmacokinetic studies, after fasting with free access to water for at least 12 h, twelve rats were randomly divided into two groups (
Blood samples, about 100
For the brain distribution studies, the remaining sixty rats were randomly divided into ten groups (
Experimental data were expressed as mean ± standard deviation (SD). Drug and Statistics 2.0 (DAS 2.0) software package (Mathematical Pharmacology Professional Committee of China, Shanghai, China) was used for estimating pharmacokinetic and brain distribution parameters by a noncompartmental method. Statistical analyses were performed with statistical software IBM SPSS Statistics 20.0 (IBM Corporation, Armonk, NY, USA). Differences between two groups were evaluated by the
The analytes and IS were found to respond best to positive ionization with the adduct ions [M + H]+, which were presented as the major peaks. Their product ion mass spectra are shown in Figure
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. The 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
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. The extraction conditions, including protein precipitation, liquid-liquid extraction, and solid-phase extraction were investigated. The 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.
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
SRM chromatograms for brigatinib (I), brigatinib-analog (II), and IS in rat plasma: (a) blank rat plasma; (b) blank plasma spiked with the analytes (1.0 ng/mL) and IS; (c) a rat plasma sample collected 1 h after oral administration of 5.0 mg/kg brigatinib; (d) a rat plasma sample collected 1 h after oral administration of 5.0 mg/kg brigatinib-analog.
The 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. The linear regression equations for brigatinib and brigatinib-analog in rat plasma were
The 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. The 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
The intrabatch and interbatch precision and accuracy of brigatinib and brigatinib-analog in rat plasma and brain homogenate (
Analytes | Matrix | Spiked conc. (ng/mL) | Intrabatch ( |
Interbatch ( | ||||
---|---|---|---|---|---|---|---|---|
Measured conc. (ng/mL) | Precision (CV%) | Accuracy (%) | Measured conc. (ng/mL) | Precision (CV%) | Accuracy (%) | |||
Brigatinib | Plasma | 1.0 | 0.92 ± 0.11 | 11.95 | 92.00 | 0.95 ± 0.10 | 10.52 | 95.00 |
2.0 | 2.03 ± 0.21 | 10.34 | 101.50 | 1.97 ± 0.18 | 9.14 | 98.50 | ||
400 | 391.88 ± 28.99 | 7.39 | 97.97 | 408.28 ± 38.34 | 9.39 | 102.07 | ||
1600 | 1514.40 ± 96.16 | 6.35 | 94.65 | 1548.96 ± 67.38 | 4.35 | 96.81 | ||
Brain homogenate | 0.5 | 0.45 ± 0.07 | 15.56 | 90.00 | 0.52 ± 0.06 | 11.53 | 104.00 | |
1.0 | 1.05 ± 0.11 | 10.47 | 105.00 | 0.96 ± 0.08 | 8.33 | 96.00 | ||
200 | 190.78 ± 19.48 | 10.21 | 95.39 | 192.07 ± 14.10 | 7.34 | 95.39 | ||
800 | 845.20 ±71.13 | 8.42 | 105.65 | 813.20 ± 51.13 | 6.29 | 101.65 | ||
Brigatinib-analog | Plasma | 1.0 | 1.07 ± 0.13 | 12.15 | 107.00 | 1.06 ± 0.11 | 10.38 | 106.00 |
2.0 | 1.87 ± 0.15 | 8.02 | 93.50 | 1.92 ± 0.14 | 7.29 | 96.00 | ||
400 | 422.46 ± 39.09 | 9.25 | 105.62 | 402.53 ± 29.17 | 7.25 | 100.63 | ||
1600 | 1537.96 ± 101.46 | 6.60 | 96.12 | 1557.67 ± 86.35 | 5.54 | 97.35 | ||
Brain homogenate | 0.5 | 0.53 ± 0.09 | 16.98 | 106.00 | 0.51 ± 0.07 | 13.73 | 102.00 | |
1.0 | 0.96 ± 0.10 | 10.42 | 96.00 | 0.97 ± 0.08 | 8.25 | 97.00 | ||
200 | 208.54 ± 16.83 | 8.07 | 104.27 | 210.19 ± 15.26 | 7.26 | 105.10 | ||
800 | 764.06 ± 54.21 | 7.09 | 95.51 | 781.64 ± 47.34 | 6.06 | 97.71 |
The 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 (
Extraction recovery and matrix effect of brigatinib and brigatinib-analog in rat plasma and brain homogenate (mean ± SD,
Analytes | Matrix | Spiked conc. (ng/mL) | Extraction recovery (%) | Matrix effect (%) | ||
---|---|---|---|---|---|---|
Mean ± SD | RSD | Mean ± SD | RSD | |||
Brigatinib | Plasma | 2.0 | 102.37 ± 9.23 | 9.02 | 92.80 ± 11.08 | 11.94 |
400 | 97.61 ± 5.55 | 5.69 | 98.62 ± 7.10 | 7.19 | ||
1600 | 93.10 ± 4.46 | 4.79 | 102.97 ± 8.82 | 8.57 | ||
Brain homogenate | 1.0 | 88.28 ± 5.13 | 5.81 | 93.78 ± 9.43 | 10.06 | |
200 | 86.81 ± 4.83 | 5.56 | 95.56 ± 8.47 | 8.86 | ||
800 | 89.74 ± 4.50 | 5.01 | 96.62 ± 8.12 | 8.40 | ||
Brigatinib-analog | Plasma | 2.0 | 112.68 ± 10.89 | 9.66 | 97.89 ± 12.09 | 12.35 |
400 | 106.42 ± 5.32 | 4.99 | 95.99 ± 6.41 | 6.68 | ||
1600 | 108.24 ± 7.72 | 7.13 | 95.94 ± 8.58 | 8.94 | ||
Brain homogenate | 1.0 | 97.43 ± 7.03 | 7.22 | 97.68 ± 7.50 | 7.68 | |
200 | 91.66 ± 5.43 | 5.92 | 102.14 ± 3.52 | 3.45 | ||
800 | 98.65 ± 8.66 | 8.78 | 94.06 ± 6.15 | 6.54 |
The stability of brigatinib and brigatinib-analog was evaluated by analysis of low and high concentration levels of QC samples under different conditions (
Stability data of brigatinib and brigatinib-analog in rat plasma and brain homogenate under various storage conditions at two QC levels (
Analytes | Matrix | Spiked conc. (ng/mL) | Storage conditions (ng/mL) | |||
---|---|---|---|---|---|---|
Ambient temperature for 6 h | −20°C for 10 d | Three freeze-thaw cycles | Autosampler at 4°C for 24 h | |||
Brigatinib | Plasma | 2.0 | 2.10 ± 0.12 | 2.05 ± 0.17 | 1.94 ± 0.09 | 1.90 ± 0.10 |
1600 | 1585.06 ± 98.31 | 1719.73 ± 107.09 | 1678.34 ± 95.87 | 1645.17 ± 118.52 | ||
Brain homogenate | 1.0 | 1.06 ± 0.08 | 0.97 ± 0.06 | 1.02 ± 0.06 | 0.96 ± 0.05 | |
800 | 788.85 ± 69.25 | 760.29 ± 48.33 | 847.44 ± 57.65 | 813.16 ± 72.23 | ||
Brigatinib-analog | Plasma | 2.0 | 2.03 ± 0.11 | 1.95 ± 0.12 | 2.01 ± 0.09 | 1.89 ± 0.12 |
1600 | 1638.54 ± 97.76 | 1705.49 ± 109.83 | 1578.36 ± 125.20 | 1582.61 ± 101.88 | ||
Brain homogenate | 1.0 | 1.04 ± 0.05 | 1.07 ± 0.11 | 0.98 ± 0.07 | 0.97 ± 0.06 | |
800 | 828.64 ± 66.54 | 846.09 ± 53.83 | 833.68 ± 56.59 | 783.28 ± 32.39 |
The 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.
No significant differences in area under the curve (AUC; 261528.73 ± 86227.28 vs. 262436.45 ± 74089.38 ng/mL·min), peak time (
Plasma concentration-time profiles of brigatinib and brigatinib-analog following single oral administration of 5 mg/kg of brigatinib and brigatinib-analog in male SD rats (
The 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. The brain distribution researches found that the brigatinib and brigatinib-analog could both penetrate the blood-brain barrier (BBB). The ability of analytes penetrating the BBB was estimated by using the ratio of average AUC(0
Brain concentration-time profiles of brigatinib and brigatinib-analog following single oral administration of 5 mg/kg of brigatinib and brigatinib-analog in male SD rats (
Pharmacokinetic parameters of brigatinib and brigatinib-analog after oral administration of 5 mg/kg of brigatinib and brigatinib-analog in male SD rats (mean ± SD,
Analytes | Units | Plasma | Brain | ||
---|---|---|---|---|---|
Brigatinib | Brigatinib-analog | Brigatinib | Brigatinib-analog | ||
AUC( |
ng/mL·min (plasma), ng/g·min (brain) | 261528.73 ± 86227.28 | 262436.45 ± 74089.38 | 58255.81 ± 13725.10 | 23196.08 ± 4932.95 |
MRT | min | 403.98 ± 12.02 | 328.55 ± 35.68 |
449.04 ± 65.44 | 379.04 ± 5.40 |
|
min | 189.41 ± 20.55 | 120.78 ± 29.74 |
319.42 ± 117.05 | 256.08 ± 40.01 |
|
min | 260.00 ± 30.98 | 260.00 ± 48.99 | 330.00 ± 176.98 | 240.00 ± 0.00 |
|
ng/mL (plasma), ng/g (brain) | 537.85 ± 185.55 | 734.41 ± 83.06 | 68.91 ± 21.57 | 45.84 ± 11.81 |
|
L/kg | 5.66 ± 1.94 | 3.43 ± 0.96 | 50.47 ± 19.42 | 119.35 ± 25.58 |
|
L/min/kg | 0.022 ± 0.010 | 0.021 ± 0.006 | 0.101 ± 0.062 | 0.325 ± 0.057 |
In conclusion, a highly sensitive, reliable, and simple LC-MS/MS method had been developed and validated for the simultaneous determination of brigatinib and brigatinib-analog in rat plasma and brain homogenate. The 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. The pharmacokinetic researches suggested that there remained some differences in pharmacokinetic characteristics between brigatinib and brigatinib-analog in rats. Moreover, compared with brigatinib-analog, brigatinib was a stronger central nervous system-penetrant. However, the future study should increase sample size to make the empirical conclusions more convincing. The above studies would lay a good foundation for the further applications of brigatinib and brigatinib-analog.
The 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.
The authors have declared no conflicts of interest.
Bo Li and Min Lu contributed equally to this work.
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.