Determination of Osimertinib, Aumolertinib, and Furmonertinib in Human Plasma for Therapeutic Drug Monitoring by UPLC-MS/MS

The third-generation epidermal growth factor receptor tyrosine kinase inhibitors (EGFR-TKIs), osimertinib, aumolertinib, and furmonertinib represent a new treatment option for patients with EGFR p.Thr790 Met (T790 M)-mutated non-small cell lung cancer (NSCLC). Currently, there are no studies reporting the simultaneous quantification of these three drugs. A simple ultra-performance liquid chromatography–tandem mass spectrometry (UPLC-MS/MS) method was developed and validated for the simultaneous quantitative determination of osimertinib, aumolertinib, and furmonertinib concentrations in human plasma, and it was applied for therapeutic drug monitoring (TDM). Plasma samples were processed using the protein precipitation method (acetonitrile). A positive ion monitoring mode was used for detecting analytes. D3-Sorafenib was utilized as the internal standard (IS), and the mobile phases were acetonitrile (containing 0.1% formic acid) and water with gradient elution on an XSelect HSS XP column (2.1 mm × 100.0 mm, 2.5 µm, Waters, Milford, MA, USA) at a flow rate of 0.5 mL·min−1. The method’s selectivity, precision (coefficient of variation of intra-day and inter-day ≤ 6.1%), accuracy (95.8–105.2%), matrix effect (92.3–106.0%), extraction recovery, and stability results were acceptable according to the guidelines. The linear ranges were 5–500 ng·mL−1, 2–500 ng·mL−1, and 0.5–200 ng·mL−1 for osimertinib, aumolertinib, and furmonertinib, respectively. The results show that the method was sensitive, reliable, and simple and that it could be successfully applied to simultaneously determine the osimertinib, aumolertinib, and furmonertinib blood concentrations in patients. These findings support using the method for TDM, potentially reducing the incidence of dosing blindness and adverse effects due to empirical dosing and inter-patient differences.


Introduction
Lung cancer, which is classified broadly as non-small cell lung cancer (NSCLC) or small cell lung cancer (SCLC), is the primary cause of cancer death worldwide and remains a major challenge [1]. NSCLC is the most common type of lung cancer, accounting for 85% of cases [2]. Therapeutic options for NSCLC have improved with the discovery of driver mutations over the past decade. Currently, molecular-targeted therapy plays a vital role in NSCLC therapies. Epidermal growth factor receptor (EGFR) mutations are the most common driver mutations in patients with advanced NSCLC, especially in Asians [3]. Molecular targeting using epidermal growth factor receptor tyrosine kinase inhibitors (EGFR-TKIs) is the first-line treatment for EGFR-mutated NSCLC [4]. However, (EGFR-TKIs) is the first-line treatment for EGFR-mutated NSCLC [4]. However, mos tients treated with first-or second-generation TKIs ultimately progress due to the e gence of the EGFR p.Thr790 Met (T790 M) point mutation. Therefore, third-gener EGFR-TKIs, including osimertinib, aumolertinib, and furmonertinib, were develop overcome this frequently acquired mutation [5].
Osimertinib (Figure 1), is a standard first-line treatment option for patients wit vanced EGFR T790 M-mutated NSCLC, effectively extending survival and improvin tients' life quality [6,7]. Subsequently, aumolertinib and furmonertinib ( Figure 1) approved in China for treating advanced EGFR T790 M-mutated NSCLC. Aumoler can effectively control metastatic brain lesions, with a confirmed survival benefit fo tients with brain metastases [8][9][10]. As the third third-generation EGFR-TKI to be keted in China, furmonertinib's unique molecular structure gives it the clinica vantages of "dual activity, high selectivity, strong tumor shrinkage, and good safety well as the ability to penetrate the blood-brain barrier. Furthermore, furmonertin gradually showing great potential for treating NSCLC [11][12][13]. However, adverse ev (AEs) and EGFR-TKI resistance can emerge, leading to dose reductions or treatmen continuation [4,14,15]. Plasma drug concentrations are closely associated with drug cacy and side effects. A correlation between the plasma concentration of some TKIs the occurrence of drug-related AEs and resistance to therapy was described by Yu [16]. Plasma concentrations of osimertinib, aumolertinib, and furmonertinib can b fected by various factors such as pathophysiology, genetic polymorphisms, patient ad ence to therapy, and interacting medications [17][18][19][20], leading to large inter-patient v bility in efficacy and AEs. Therefore, it is necessary to monitor disease response plasma drug concentrations to improve patient outcomes [21]. Therapeutic drug monitoring (TDM) is an important technique for formulating ing regimens for drugs with strong toxic effects, and great individual differences m ured by drug concentrations in biological samples can identify inter-patient differe and improve the therapeutic effects of drugs while reducing AEs [22][23][24]. Several stu have also shown that TDM can improve the therapeutic efficacy of TKIs, supportin tional clinical use [25][26][27][28]. The ultra-performance liquid chromatography-tandem spectrometry (UPLC-MS/MS) method is a standard analytical tool for TDM and is w used in clinical practice for precise treatment with targeted oral drugs. Although se LC-MS/MS methods have been developed for osimertinib quantification in human pl few methods [29,30] have been designed for determining the blood levels of aumoler and furmonertinib. Furthermore, there are currently no available methods for the si taneous quantification of osimertinib, aumolertinib, and furmonertinib in human pla We developed a rapid, sensitive, and efficient UPLC-MS/MS method to simul ously determine osimertinib, aumolertinib, and furmonertinib concentrations in hu Therapeutic drug monitoring (TDM) is an important technique for formulating dosing regimens for drugs with strong toxic effects, and great individual differences measured by drug concentrations in biological samples can identify inter-patient differences and improve the therapeutic effects of drugs while reducing AEs [22][23][24]. Several studies have also shown that TDM can improve the therapeutic efficacy of TKIs, supporting rational clinical use [25][26][27][28]. The ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) method is a standard analytical tool for TDM and is widely used in clinical practice for precise treatment with targeted oral drugs. Although several LC-MS/MS methods have been developed for osimertinib quantification in human plasma, few methods [29,30] have been designed for determining the blood levels of aumolertinib and furmonertinib. Furthermore, there are currently no available methods for the simultaneous quantification of osimertinib, aumolertinib, and furmonertinib in human plasma.
We developed a rapid, sensitive, and efficient UPLC-MS/MS method to simultaneously determine osimertinib, aumolertinib, and furmonertinib concentrations in human Molecules 2022, 27, 4474 3 of 11 plasma. A protein precipitation method was used for sample pretreatment, and gradient elution was used to separate the analytes, which effectively reduced the measurement time and improved the detection efficiency for the target drugs. A quantitative analysis of patient plasma samples was performed to provide a basis for rational clinical drug use.

Method Development and Optimization
A UPLC-MS/MS method was developed for the simultaneous quantification of osimertinib, aumolertinib, and furmonertinib concentrations. The MS/MS parameters, mobile phase, and gradient elution steps used in this study were optimized to meet the requirements for the simultaneous detection of three drugs in human plasma. The runtime was 4.1 min under gradient elution, where acetonitrile was used as the organic phase. Formic acid (0.1%) was added to the organic phase to obtain distinct symmetric peaks and minimal background noise, and the final choice was acetonitrile (containing 0.1% formic acid) and water. The optimization of the mass spectrometry conditions revealed a higher response for the analytes in the positive ion mode. [M + H] + was used as the parent ion for analytes because it had the best response. The target ion transitions obtained after the screening of osimertinib, aumolertinib, furmonertinib, and IS were as follows: 500.1→72.2, 526.1→72.2, 569.1→72.1, and 468.2→255.4 ( Figure 2). Other MS conditions, including the declustering potential (DP), collision energy (CE), and ion source temperature (TEM), were also optimized. plasma. A protein precipitation method was used for sample pretreatment, and grad elution was used to separate the analytes, which effectively reduced the measurem time and improved the detection efficiency for the target drugs. A quantitative analys patient plasma samples was performed to provide a basis for rational clinical drug u

Method Development and Optimization
A UPLC-MS/MS method was developed for the simultaneous quantification of mertinib, aumolertinib, and furmonertinib concentrations. The MS/MS parameters, bile phase, and gradient elution steps used in this study were optimized to meet th quirements for the simultaneous detection of three drugs in human plasma. The run was 4.1 min under gradient elution, where acetonitrile was used as the organic ph Formic acid (0.1%) was added to the organic phase to obtain distinct symmetric peaks minimal background noise, and the final choice was acetonitrile (containing 0.1% fo acid) and water. The optimization of the mass spectrometry conditions revealed a hi response for the analytes in the positive ion mode. [M + H] + was used as the parent io analytes because it had the best response. The target ion transitions obtained afte screening of osimertinib, aumolertinib, furmonertinib, and IS were as follows: 500.1→ 526.1→72.2, 569.1→72.1, and 468.2→255.4 ( Figure 2). Other MS conditions, including declustering potential (DP), collision energy (CE), and ion source temperature (T were also optimized. Isotope-labeled internal standards are most commonly used in tandem mass s trometry to eliminate errors due to matrix interference and differential ionization pro ties of the analytes. In previous studies [29,30], the IS used to detect aumolertinib furmonertinib was a deuterated internal standard. However, deuterated aumolertinib furmonertinib were not readily available, and d3-sorafenib was selected as the inte standard (IS) in this study. No significant matrix effects were observed at the reten times of the analytes. Compared to liquid-liquid extraction and solid-phase extrac the protein precipitation method has the advantages of simplicity, speed, low cost, reduced environmental pollution and is more suitable for TDM. Therefore, aceton was selected as the protein precipitant in this study.

Analytical Method Validation
The retention times for osimertinib, aumolertinib, furmonertinib, and IS were min, 1.72 min, 1.80 min, and 2.34 min, respectively. At the retention times of the ana Isotope-labeled internal standards are most commonly used in tandem mass spectrometry to eliminate errors due to matrix interference and differential ionization properties of the analytes. In previous studies [29,30], the IS used to detect aumolertinib and furmonertinib was a deuterated internal standard. However, deuterated aumolertinib and furmonertinib were not readily available, and d 3 -sorafenib was selected as the internal standard (IS) in this study. No significant matrix effects were observed at the retention times of the analytes. Compared to liquid-liquid extraction and solid-phase extraction, the protein precipitation method has the advantages of simplicity, speed, low cost, and reduced environmental pollution and is more suitable for TDM. Therefore, acetonitrile was selected as the protein precipitant in this study.

Selectivity
The retention times for osimertinib, aumolertinib, furmonertinib, and IS were 1.29 min, 1.72 min, 1.80 min, and 2.34 min, respectively. At the retention times of the analytes and the IS, endogenous substances in the plasma did not interfere with the detection of each analyte, demonstrating the good selectivity and specificity of the method ( Figure 3).
Molecules 2022, 27, x FOR PEER REVIEW 4 and the IS, endogenous substances in the plasma did not interfere with the detectio each analyte, demonstrating the good selectivity and specificity of the method (Figur

Precision and Accuracy
The intra-day precision, inter-day precision, and accuracy of the quality control ( samples and LLOQ samples for all analytes met the requirements, and the results within acceptable limits, as shown in Table 1.

Precision and Accuracy
The intra-day precision, inter-day precision, and accuracy of the quality control (QC) samples and LLOQ samples for all analytes met the requirements, and the results are within acceptable limits, as shown in Table 1.

Matrix Effect and Extraction Recovery
The validation results for the matrix effect and extraction recovery for each compound are shown in Table 2. The results indicate that the endogenous substances did not interfere with the analyte detection.

Stability
The stability of each analyte was examined under various storage conditions using QC plasma samples for osimertinib, aumolertinib, and furmonertinib. The results indicate the acceptable stability of the QC samples for all analytes under the storage conditions tested (Table 3).

Carry-Over
The carry-over effect was assessed by analyzing a blank matrix sample after an upper limit of quantification (ULOQ) sample had been injected. At the retention time of the analyte, the peak areas of interfering peaks in the blank matrix sample were less than 20% of that of the LLOQ sample. Furthermore, there were no significant interfering peaks at the retention time of the IS, indicating that the high concentration sample had no carry-over effect on the determination of the low concentration sample.

Clinical Application
The validated method was successfully used in our laboratory to measure the plasma drug concentration at steady states of osimertinib (n = 10) and aumolertinib (n = 2) in patients with NSCLC (Table 4 and Figure 4).

Carry-Over
The carry-over effect was assessed by analyzing a blank matrix sample after an up limit of quantification (ULOQ) sample had been injected. At the retention time of the alyte, the peak areas of interfering peaks in the blank matrix sample were less than of that of the LLOQ sample. Furthermore, there were no significant interfering peak the retention time of the IS, indicating that the high concentration sample had no ca over effect on the determination of the low concentration sample.

Clinical Application
The validated method was successfully used in our laboratory to measure the pla drug concentration at steady states of osimertinib (n = 10) and aumolertinib (n = 2) in tients with NSCLC (Table 4 and Figure 4).  Consistent with previous publications [28,31], the minimum drug concentrat (Cmin) of osimertinib at a steady state were highly variable (6.19-380 ng/mL), and the m Cmin was 139.98 ng/mL at the steady state. Compared to the methods previously repor our newly developed method was more simple and convenient.
There are limited data on the Cmin of aumolertinib and furmonertinib in patients w NSCLC. Phase I clinical trial data show that steady-state Cmin is 193 ng/mL after the ministration of 110 mg of aumolertinib, and 29.1 ng/mL after the administration of 80 of furmonertinib [9,11]. In our study, plasma concentrations at a steady state for two tients (two samples) treated with 110 mg aumolertinib once daily were analyzed. Pla samples from patients treated with furmonertinib were not available. The mean Cm aumolertinib was 155.5 ng/mL, which is consistent with the published clinical trial da Brown et al. [32] did not identify a relationship between osimertinib exposure efficacy, but a correlation between exposure and safety endpoints was observed. Du the small number of clinical plasma samples, the relationships between the plasma c centrations of the three drugs and their efficacy and side effects were not establishe Consistent with previous publications [28,31], the minimum drug concentrations (C min ) of osimertinib at a steady state were highly variable (6.19-380 ng/mL), and the mean C min was 139.98 ng/mL at the steady state. Compared to the methods previously reported, our newly developed method was more simple and convenient.
There are limited data on the C min of aumolertinib and furmonertinib in patients with NSCLC. Phase I clinical trial data show that steady-state Cmin is 193 ng/mL after the administration of 110 mg of aumolertinib, and 29.1 ng/mL after the administration of 80 mg of furmonertinib [9,11]. In our study, plasma concentrations at a steady state for two patients (two samples) treated with 110 mg aumolertinib once daily were analyzed. Plasma samples from patients treated with furmonertinib were not available. The mean C min of aumolertinib was 155.5 ng/mL, which is consistent with the published clinical trial data.
Brown et al. [32] did not identify a relationship between osimertinib exposure and efficacy, but a correlation between exposure and safety endpoints was observed. Due to the small number of clinical plasma samples, the relationships between the plasma concentrations of the three drugs and their efficacy and side effects were not established in our study.
The collection of clinical samples from patients treated with osimertinib, aumolertinib, and furmonertinib is ongoing. There are some active metabolites of osimertinib, aumolertinib, and furmonertinib present in plasma. However, due to their low concentration [33] and difficulty in obtaining reference standards, they were not analyzed in this study, which is also a limitation of our study. The method developed in this study could be used to monitor drug plasma concentrations and the associated disease response to achieve appropriate therapeutic strategies.

Plasma Sample Preparation
Plasma samples were processed using the protein precipitation method. Ten microliters of IS (200 ng·mL −1 ) were added to 100 µL of plasma sample (including calibration curve or QC samples (10 µL of calibration standard solution or QC working solution were added to 90 µL of blank plasma)). Then, 300 µL of acetonitrile were added, vortexed for 1 min, and centrifuged at 12,000 rpm for 10 min. The supernatant (100 µL) was added to 300 µL of 50% acetonitrile, vortexed and mixed, and finally transferred to an autosampler vial for sample analysis.

Method Validation
The method was comprehensively validated for selectivity, calibration curve linearity, the LLOQ, precision and accuracy, matrix effect, extraction recovery, stability, and carryover according to the US FDA [34] and Chinese Pharmacopoeia (2020) Guidelines for the Validation of Bioanalytical Methods.

Selectivity
The method's selectivity was determined by assessing the interference of other components in plasma. Plasma samples containing 2 ng·mL −1 aumolertinib were prepared by sequentially adding the aumolertinib working solution (20 ng·mL −1 ) to blank plasma from different sources (n = 6). The samples were processed as described in Section 3.4. Plasma samples containing osimertinib and furmonertinib were also prepared using this procedure, and the plasma was then analyzed. In the absence of interference, the peak area of the analyte in the blank plasma should be less than 20% of the LLOQ and 5% of the IS within the retention time.

Calibration Curve and LLOQ
Using the concentration of the analyte as the horizontal coordinate (x), the peak area ratio of the analyte to the IS as the vertical coordinate (y), and 1/x 2 as the weighting factor, a weighted least squares method was used to obtain the regression equation and generate the calibration curve. The linear ranges of the standard curves for osimertinib, aumolertinib, and furmonertinib were 5-500 ng·mL −1 , 2-500 ng·mL −1 , and 0.5-200 ng·mL −1 , respectively. The difference between the back-calculated and the nominal concentrations for each standard in the calibration curve had an acceptable range (<15%). For LLOQ, the difference should be less than 20%.

Precision and Accuracy
The precision and accuracy were assessed by measuring low, medium, and high concentrations of QC (LQC, MQC, HQC) samples and LLOQ samples. Intra-day and interday precision and accuracy were calculated by repeating the measurements six times a day for three consecutive days for samples at each concentration level. Precision was expressed by calculating the relative standard deviation (RSD) of the samples from six parallel measurements. Accuracy was expressed by the relative error (RE) of the samples. The RSD and RE were within ±15% for the QC samples and within ±20% for the LLOQ samples.

Matrix Effect and Extraction Recovery
The matrix effect for the analyte was determined by comparing the analyte peak area in the blank plasma samples from six different donors at LQC and HQC levels (n = 6 for each level) with the analyte peak area in pure solution. The extraction recovery was assessed by comparing the analyte peak area in extracted plasma samples at LQC, MQC, and HQC levels (n = 6 for each level), with the analyte peak area of a blank plasma extract spiked at the same level.

Stability
The stability of the samples under different storage or handling conditions was assessed by analyzing the QC samples at different levels (LQC, MQC, HQC; n = 6). The short-term stability was assessed after placing the plasma samples at room temperature for 8 h. Quality control samples were stored at −20 • C for 7 d to assess the long-term stability. We also examined the stability of the samples stored in a refrigerator at 4 • C for 24 h. The stability of the processed samples was assessed by storing the samples in an autosampler at 4 • C for 24 h. The samples stored at −20 • C were thawed at room temperature, and three freeze-thaw cycles were performed to investigate the freeze-thaw stability of the samples.

Carry-Over
The carry-over of this method was investigated by sequentially injecting the LLOQ and the ULOQ samples, followed by a blank biological matrix sample. The area of interfering peaks at the retention time of the analyte in the blank plasma sample should be less than 20% of the LLOQ and 5% of the IS peak areas.

Clinical Samples Analysis
All the experimental procedures were approved by the Ethics Committee of Hebei General Hospital (No. 2022094).
Plasma samples from patients who had been administered osimertinib, aumolertinib, or furmonertinib for at least two weeks and were within half an hour before readministration were analyzed to demonstrate the applicability of the assay. The clinical blood samples were residual blood samples obtained from patients for other routine clinical measurements at Hebei General Hospital. Blood was collected in EDTA-containing anticoagulation tubes, and the supernatant was separated by centrifugation at 3000 rpm for 10 min at 4 • C. Then, the supernatant was transferred to 1.5 mL EP tubes and stored at −20 • C until analysis.

Conclusions
We have developed a simple, rapid, and sensitive UPLC/MS/MS method to simultaneously determine osimertinib, aumolertinib, and furmonertinib concentrations in human plasma. The validated method may also be a useful tool for the TDM of the third-generation EGFR-TKIs for NSCLC patients in clinical practice.

Informed Consent Statement:
The need for informed consent from included individuals was waived by the Ethics Committee as clinical routine blood residual samples were used in this study.