Osimertinib is a novel oral, potent, and irreversible epidermal growth factor receptor tyrosine kinase inhibitor (EGFR-TKI) for treatment of advanced T790M mutation-positive advanced non-small cell lung cancer, which is commonly combined with ginsenoside Rg3 in clinic to enhance the efficacy and minimize adverse reactions. In the present study, a highly sensitive UPLC-MS/MS method was established and validated for analysis of osimertinib in rat plasma according to US FDA guideline. Separation was performed on a C18 (2.1 × 50 mm, 2.6
Osimertinib (AZD9291), the first third-generation oral potent and irreversible epidermal growth factor receptor tyrosine kinase inhibitor (EGFR-TKI), was approved by US-FDA and NMPA for first-line treatment of advanced non-small cell lung cancer (NSCLC) patients with acquired metastatic EGFR T790M mutation [
Ginsenoside Rg3, the compound extracted from the Chinese herb Panax ginseng, proved to inhibit the growth, invasion, and metastasis of several kinds of malignancies, including lung cancer, breast cancer, ovarian cancer, glioma, and leukemia, and enhance the efficacy of chemotherapy. Kim et al. reported that Shenyi capsule (the main active ingredient is ginsenoside Rg3) could improve the life span of NSCLC patients by improving the immune function and antitumor angiogenesis [
An appropriate analytical method should be established for evaluating the drug interaction between Rg3 and osimertinib. However, to the best of our knowledge, there were only two published articles about liquid chromatography-tandem mass spectrometry (LC-MS/MS) methods for determination of osimertinib in rat plasma [
Osimertinib (purity ≥ 99.95%) was purchased from MedChemExpress Co., Ltd. (Shanghai, China). Nilotinib (purity ≥ 99%) was procured from Aladdin Co., Ltd. (Shanghai). Shenyi capsule (mainly included 10 mg ginsenoside Rg3 in one capsule) was supplied from Yatai Pharmaceuticals Co., Ltd. (Jiutai, China). The chemical structures of the analytes are depicted in Figure
The chemical structures of osimertinib (a) and nilotinib (IS) (b).
Male Sprague-Dawley rats, weighing 200–220 g, were obtained from Beijing Vital River Laboratory Animal Technology (Beijing, China). The rats were housed in an environmentally controlled breeding room (12 h light/dark cycle) for three days before the experiment, and the standard laboratory food and water were available
Dissolving accurately weighed amounts of standard osimertinib and nilotinib in methanol to obtain the stock solutions in a final concentration of 2 mg/mL. To prepare the working solutions, stock solutions were diluted with ACN/water (1/1, V/V). The calibration standard samples were prepared by spiking the corresponding working solutions with blank rat plasma. Final calibration standard concentrations for osimertinib were 400, 300, 200, 100, 50, 20, 5, and 1 ng/mL, respectively. In a similar way, LLOQ and QC samples were prepared in diluting working solutions with rat plasma at four concentration levels: 350 ng/mL (high quality control (HQC)), 30 ng/mL (medium quality control (MQC)), 3 ng/mL (low quality control (LQC)), and 1 ng/mL (lower limit of quantification (LLOQ)). All stock and working solutions were stored at −80°C until further analysis.
Samples were extracted from rat plasma by protein precipitation. For this method, a total of 50
Osimertinib and IS were analyzed by an Exion LC Analytical System (AB Sciex, USA) coupled with a Qtrap 4500 mass spectrometer equipped with Turbo Ion Spray Interface (AB Sciex, USA). Separation of osimertinib and IS was achieved on a Phenomenex HPLC Kinetex C18 column (2.1 × 50 mm, 2.6
The Qtrap 4500 mass spectrometer equipped with Turbo Ion Spray interface operating in positive ESI mode (AB Sciex, USA) was selected for mass spectrometric detection. The multiple reaction monitoring (MRM) mode was acquired, and the MS spectrometry parameters were defined as follows: source temperature 500°C; ion spray voltage 4500 V; nebulizer gas (gas1) 50 psi; heater gas (gas2) 50 psi; curtain gas 40 psi; a low collision gas. The dwell time for osimertinib and nilotinib was 100 ms. The mass-dependent parameters are summarized in Table
General mass spectrometric settings for the osimertinib and internal standards.
Analytes | ESI | RT (min) | DP (V) | EP (V) | CE (V) | CXP (V) | |
---|---|---|---|---|---|---|---|
Osimertinib | + | 2.51 | 95 | 10 | 34 | 16 | |
Nilotinib (IS) | + | 2.90 | 150 | 10 | 40 | 7 |
According to the guidelines of the US Food and Drug Administration (FDA) [
To explore the selectivity and specificity of this method, six individual blank rat plasma samples, blank rat plasma samples spiked with osimertinib and IS at LLOQ level, and actual rat plasma samples after oral administration of osimertinib were analyzed.
The linearity, expressed by calibration curves with the correlation coefficient (
Four different concentration levels of QC samples (1, 3, 30, and 350 ng/mL for LLOQ, LQC, MQC, and HQC, resp.) with six replicates for osimertinib were determined to evaluate the precision (intraday and interday) and accuracy of this method. The intraday experiment was assayed in one run within one day, and the interday experiment was assessed in three analytical runs on three separate days. The intraday and interday precision were assessed by calculating the relative standard deviation (RSD) and accuracies, which was required to be within ±15% of the nominal concentrations for LQC, MQC, and HQC samples and should be less than 20% RSD for LLOQ. Accuracy was calculated by analyzing the averaged measurements to normal values, which was expressed in relative error percentage: [(calculated concentration − true concentration/true concentration)
The matrix effect of osimertinib was investigated by matrix factor, a peak area ratio of the analyte/IS with three QC concentrations in the presence of matrix ions (rat plasma) to those in the absence of matrix at equivalent concentrations. The RSD% of the matrix effect should less than 15%. The recovery was assessed by comparing the peak areas obtained from extracted QC samples with those in the mobile phase at the same concentrations and expressed as percentage. The matrix effect and recovery of IS were evaluated in the similar method at a concentration of 600 ng/mL in plasma.
The rats were randomly divided into two groups (six rats for each group). Osimertinib was dissolved and suspended by 1% carboxymethylcellulose sodium solution. The Rg3 powder in Shenyi capsule was dissolved in saline. In the first period, ginsenoside Rg3 (5 mg/kg) was orally administered to the rats in group I for seven days, and the rats in the other group were orally treated with normal saline for seven days. In the second period, the rats were fasted overnight with free access to water before the experiment. After overnight fasting, ginsenoside Rg3 (5 mg/kg) plus osimertinib (10 mg/kg) was orally administered to the rats in group I, and the rats received oral ginsenoside Rg3 30 min prior to osimertinib at the eighth day. Rats in group II were only orally treated with osimertinib (10 mg/kg) at the eighth day. Blood samples (each 100
The pharmacokinetic parameters, including the maximal plasma concentration (
All data were presented as the mean ± SD. Statistical differences analyses between the mean values were performed in SPSS software version 11.5 (SPSS, Chicago, IL, USA) and analyzed for significance by a nonpaired two-tailed Student's t-test.
In order to achieve high sensitivity and better response, the positive ionization and multiple reaction monitoring scan mode was applied and optimized by a systematic approach. The precursor and product ions were determined by directly injecting standard solution of osimertinib and IS to the mass spectrometer (Figure
Product ion mass spectra of osimertinib (a) and nilotinib (b) in positive mode and their proposed fragmentation patterns.
To optimize the appropriate chromatographic conditions for separation, commercially available columns and various mobile phases (various proportions and gradients) were evaluated for their chromatographic behavior and the ionization response.
The results indicated that Phenomenex HPLC Kinetex C18 column (2.1 × 50 mm, 2.6
Various extraction was evaluated to achieve a maximum extraction efficiency of osimertinib and IS in rat plasma. The solid-phase extraction method was unnecessary for its complication. Poor recovery was observed in liquid-liquid extraction method with
There was no significant chromatographic interference peaks from endogenous substances at the retention time with the osimertinib and IS in rat plasma. The responses of osimertinib in blank rat plasma samples were all <20% of the LLOQ response, and the blank IS responses were below 1% of the normal response. The typical chromatograms of blank rat plasma, blank plasma sample spiked with osimertinib and IS at LLOQ level, and rat plasma sample after administration were shown in Figure
Typical MRM chromatograms of osimertinib and nilotinib (IS). (a) Blank plasma matrix sample; (b) blank plasma matrix sample spiked with the analytes (at medium concentrations); (c) real plasma sample obtained from rat after intravenous administration of osimertinib.
Good linearity of the calibration curve of osimertinib, generated by linear regression of peak area ratios against concentrations with a weighting factor of 1/
Calibration standards for osimertinib in rat plasma (linear weighted 1/
Analytes | Item | Nominal standards concentrations (ng/mL) | QCs (ng/mL) | ||||||||||
---|---|---|---|---|---|---|---|---|---|---|---|---|---|
S1 (1) | S2 (5) | S3 (20) | S4 (50) | S5 (100) | S6 (200) | S7 (300) | S8 (400) | L (3) | M (30) | H (350) | |||
Osimertinib | Mean | 0.98 | 4.85 | 20.61 | 48.82 | 93.82 | 218.05 | 293.07 | 405.60 | 2.96 | 31.29 | 361.65 | 0.9972 |
RSD (%) | 5.61 | 6.06 | 9.68 | 3.39 | 5.95 | 6.84 | 0.79 | 3.65 | 8.48 | 1.71 | 4.75 | 0.12 | |
RE (%) | −2.31 | −3.01 | 3.05 | −2.36 | −6.18 | 9.02 | −2.31 | 1.27 | −1.27 | 4.28 | 3.33 |
The intra- and interday precision and accuracy for the determination of osimertinib at LLOQ and three QC levels in rat plasmas on three consecutive days are summarized in Table
Intraday and interday precision and accuracy of osimertinib in rat plasma.
Analytes | Spiked concentration (ng/mL) | Intraday ( | Interday ( | ||||
---|---|---|---|---|---|---|---|
Mean ± SD (ng/mL) | RSD (%) | Accuracy (%) | Mean ± SD (ng/mL) | RSD (%) | Accuracy (%) | ||
Osimertinib | 1 (LLOQ) | 1.02 ± 0.11 | 10.4 | 102.5 | 0.99 ± 0.10 | 10.3 | 98.5 |
3 (QC-L) | 2.94 ± 0.20 | 6.9 | 98.1 | 2.91 ± 0.20 | 6.8 | 97.0 | |
30 (QC-M) | 31.0 ± 0.5 | 1.7 | 103.2 | 30.3 ± 1.6 | 5.2 | 101.1 | |
350 (QC-H) | 368 ± 11 | 3.1 | 105.1 | 365 ± 13 | 3.5 | 104.3 |
The ratio of the peak area in the presence of matrix to the peak area in absence of matrix was determined as matrix factor. The IS-normalized osimertinib was calculated by dividing the osimertinib of the analyte by the osimertinib of the IS. As shown in Table
Matrix effect and extraction recovery of osimertinib in rat plasma (
Analytes | Spiked concentration (ng/mL) | Matrix effect | Extraction recovery | ||
---|---|---|---|---|---|
Mean ± SD (%) | RSD (%) | Mean ± SD (%) | RSD (%) | ||
Osimertinib | 3 (QC-L) | 105 ± 7 | 7.0 | 108 ± 2 | 1.7 |
30 (QC-M) | 109 ± 6 | 5.7 | 100 ± 7 | 7.1 | |
350 (QC-H) | 114 ± 1 | 0.7 | 110 ± 8 | 7.6 | |
IS | 600 | — | — | 99.0 ± 7.9 | 8.0 |
The stability of osimertinib at three QC concentrations under four different storage conditions is described in Table
Stability of osimertinib in rat plasma under various storage conditions (
Spiked concentration (ng/mL) | Short-term (room temperature for 6 h) | Autosampler (4°C for 24 h) | Freeze-thawing (−80°C for 3 cycles) | Long-term (−80°C for 28 days) | ||||||||
---|---|---|---|---|---|---|---|---|---|---|---|---|
Mean ± SD (ng/mL) | RSD (%) | RE (%) | Mean ± SD (ng/mL) | RSD (%) | RE (%) | Mean ± SD (ng/mL) | RSD (%) | RE (%) | Mean ± SD (ng/mL) | RSD (%) | RE (%) | |
3 | 3.20 ± 0.14 | 4.2 | 6.8 | 3.02 ± 0.01 | 0.4 | 0.6 | 3.03 ± 0.02 | 0.8 | 1.0 | 2.96 ± 0.24 | 8.2 | −1.5 |
30 | 30.1 ± 1.0 | 3.1 | 0.4 | 28.7 ± 0.8 | 2.7 | −4.2 | 30.1 ± 1.0 | 3.1 | 0.4 | 32.7 ± 0.8 | 2.3 | 9.1 |
350 | 341 ± 3 | 0.8 | −2.7 | 359 ± 26 | 7.1 | 2.5 | 349 ± 6 | 1.6 | −0.3 | 359 ± 7 | 2.0 | 2.4 |
The current fully validated UPLC-MS/MS method was successfully applied for determining the concentration of osimertinib in rat plasma after oral administration of osimertinib (10 mg/kg) to male rats in the presence or absence of Rg3 (5 mg/kg) for seven days. The mean plasma concentration–time curves of osimertinib in rats are shown in Figure
Mean plasma concentration–time profiles of osimertinib in male rats (each point represents mean ± SD). (A) Mean plasma concentration–time profiles of osimertinib after intravenous administration at a single dose of 10 mg/kg in male rats (
Pharmacokinetic parameters of osimertinib following oral administration (10 mg/kg) to male rats in the presence or absence of Rg3 (5 mg/kg, 7 days,
PK parameter | Osimertinib alone | Rg3 + Osimertinib |
---|---|---|
0.317 ± 0.138 | 0.297 ± 0.122 | |
3.33 ± 0.82 | 2.60 ± 0.55 | |
4.46 ± 0.94 | 5.08 ± 1.03 | |
AUC0- | 2.82 ± 1.20 | 2.60 ± 0.84 |
AUC0-∞ (h· | 2.90 ± 1.21 | 2.72 ± 0.90 |
MRT0-t (h) | 6.88 ± 0.62 | 7.18 ± 0.75 |
CL/F (L/h/kg) | 19.8 ± 8.1 | 20.0 ± 6.2 |
Vz/F (L/kg) | 130 ± 58 | 146 ± 54 |
Values are mean ± SD.
The pharmacokinetic data showed that osimertinib was slowly absorbed at the highest plasma concentration in a long time (
Small-molecule tyrosine kinase inhibitors (TKIs) that target the EGFR are commonly used in combination with Chinese herbal medicines (e.g., ginseng, astragalus, and
On coadministering with Rg3 (5 mg/kg) for seven days,
This validated UPLC-MS/MS method and pharmacokinetic study may prove to be of great significance for future investigations when conducting human clinical drug interaction trials on Rg3 and osimertinib.
In conclusion, a sensitive, rapid, and selective UPLC-MS/MS method was developed and validated for quantification of osimertinib in rat plasma, within a linear range of 1–400 ng/mL. This method was successfully applied to the pharmacokinetic study of osimertinib in rats across different administrations. This is the first study that discloses the effect of Rg3 on the pharmacokinetic profiles of osimertinib in rats at a clinical dosage level. Pharmacokinetic properties demonstrated that osimertinib exhibited a slow absorption and moderate-rate elimination in rats following oral administration of osimertinib (10 mg/kg). Coadministration with Rg3 (5 mg/kg) for seven days may have no obvious effects on the pharmacokinetics of osimertinib in rats. This study provides the theoretical foundation for the concomitant medications of Rg3 and osimertinib such that Rg3 could improve the life span, quality of life, and survival rate of NSCLC patients.
Non-small cell lung cancer
Epidermal growth factor receptor tyrosine kinase inhibitor
Ultra-performance liquid chromatography-tandem mass spectrometry
Collision cell exit potential
Declustering potential
Entrance potential
Collision energy
Internal standard
Multiple reaction monitoring
Electrospray ionization
Lower limit of quantification
Matrix effect
Sodium carboxymethylcellulose
Quality control
Upper limit of quantification
Relative standard deviation
Relative error
Acetonitrile.
The data used to support the findings of this study are included within the article.
Zhenzhen Ying and Jingyao Wei are co-first authors.
The authors declare no conflicts of interest.
This study was financially supported by the National Science and Technology Major Project of China (2020ZX09201009), the grants from the National Natural Science Foundation of China (31870809), the Henan Science and Technology Foundation (172102310145) and the Youth Innovation Fund of the First Affiliated Hospital of Zhengzhou University (YNQN2017202).