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. 2024 Jan 24;46(3):332–343. doi: 10.1097/FTD.0000000000001157

A Validated Assay to Quantify Osimertinib and Its Metabolites, AZ5104 and AZ7550, from Microsampled Dried Blood Spots and Plasma

Bharat Venkatesh *, Alex Yuile †, Matthew J McKay *, Sathya Narayanan †, Helen Wheeler †, Malinda Itchins †, Nick Pavlakis †, Stephen J Clarke †, Mark P Molloy *,✉
PMCID: PMC11078286  PMID: 38263583

Abstract

Background:

Osimertinib is an oral small-molecule tyrosine kinase receptor inhibitor used to treat non–small cell lung cancer (NSCLC) with a sensitizing epidermal growth factor receptor mutation. Patients may experience drug toxicity and require dose deescalation. The study aimed to quantitate osimertinib and its 2 active metabolites, AZ5104 and AZ7550, in microsampled dried blood spots (DBS) collected from patients with NSCLC using a hemaPEN device and compare them with plasma drug levels.

Methods:

A 6-min ultrahigh-performance liquid chromatography–tandem mass spectrometry method was developed and validated using plasma and DBS. The accuracy, selectivity, matrix effect, recovery, and stability were assessed using bioanalytical validation criteria. The hematocrit effect was investigated in DBS. Drug levels were measured in 15 patients with NSCLC, and the Bland–Altman method was used to compare measurements between plasma and DBS.

Results:

The validated assay determined accurate and precise quantities, respectively, for osimertinib in both plasma (93.2%–99.3%; 0.2%–2.3%) and DBS (96.7%–99.6%; 0.5%–10.3%) over a concentration of 1–729 ng/mL. The osimertinib metabolites, AZ5104 and AZ7550, were similarly validated in accordance with bioanalytical guidelines. For 30%–60% patient hematocrit, no hematocrit bias was observed with DBS for all analytes. The Bland–Altman method showed high concordance between plasma and DBS analyte levels. Stability experiments revealed that osimertinib and its metabolites were poorly stable in plasma at room temperature, whereas all analytes were stable in DBS for 10 days at room temperature.

Conclusions:

The measurement of osimertinib, AZ5104, and AZ7550 from hemaPEN microsampled DBS is a convenient and reliable approach for therapeutic drug monitoring that produces measurements consistent with plasma drug levels.

Key Words: tyrosine kinase inhibitors, therapeutic drug monitoring, liquid chromatography–tandem mass spectrometry, osimertinib, non-small cell lung cancer

INTRODUCTION

Treatment with tyrosine kinase inhibitors (TKIs) is the current standard of care for patients with advanced non–small cell lung cancer (NSCLC) harboring a sensitizing epidermal growth factor receptor (EGFR) mutation. First-generation TKIs, gefitinib and erlotinib, demonstrated extended progression-free survival compared with platinum chemotherapy.1–5 A meta-analysis involving patients from 6 randomized trials who had not received any prior treatment showed a progression-free survival of 11.0 months with EGFR-TKIs (gefitinib or erlotinib) compared with 6 months with chemotherapy.6 Second-generation ErbB family-targeting agents, afatinib and dacomitinib, showed improved efficacy relative to both standard platinum-based chemotherapy and first-generation EGFR-TKIs. Although treatment with EGFR-TKIs has benefited many patients with activating EGFR mutations, almost all patients, who initially benefit from this treatment, develop acquired resistance.7 Since 2005, several mutations have been associated with TKI resistance, and among them, the gatekeeper T790M is by far the most studied mutation. More than 50% of the patients who develop this resistance have the T790M mutation.8

Osimertinib is an approved, oral, third-generation, irreversible EGFR-TKI that selectively inhibits both EGFR-TKI–sensitizing and EGFR T790M resistance mutations by binding to cysteine 797 in the ATP-binding site of EGFR, exhibiting 200× greater potency toward mutated EGFR/T790M than wild-type EGFR.9–12 Several studies have demonstrated that osimertinib improves overall survival when used as a first-line anti-EGFR therapy in advanced NSCLC with a sensitizing mutation, irrespective of T790M status. Preclinical data suggest that osimertinib is principally metabolized by cytochrome P450 (CYP3A4) and produces at least 2 circulating active metabolites: AZ5104 and AZ7550.13,14 Patients (20%–42%) receiving osimertinib develop grade 3 or higher toxicity, which may require hospitalization and dose deescalation, which cannot be predicted.15–17 Agema et al18 recently proposed a toxic limit for osimertinib plasma concentration, that is, 259 ng/mL, paving the way for therapeutic drug monitoring (TDM).

Several new devices for blood microsampling have increased interest for TDM because they allow a patient-friendly, do-it-yourself approach for sample collection.19,20 One such device is the hemaPEN (Trajan, Melbourne, Australia), which collects precise blood volumes and deposits them onto prepunched filter paper discs (3.5-mm diameter; PE 226 filter paper), creating a dried blood spot (DBS).21,22 This highly controlled approach obviates the blood hematocrit effect commonly observed in conventional DBS applied to cards.23,24 The hemaPEN device can be used with finger-prick blood drops, and it has 4 integrated 2.74-μL microcapillaries that are filled by capillary action. Upon engaging the device back into its base, the capillaries are emptied, and the blood is deposited onto 4 prepunched paper discs. An integrated desiccant aids in sample drying and storage. Use of hemaPEN includes DBS measurement of acetaminophen,25 tacrolimus,26 lysosomal enzymes,27 and biological acids and lipids.28 An alternative microsampling device based on polymer volumetric adsorptive microsampling demonstrated reliable blood-based drug quantitation for several TKIs, including osimertinib.29 However, real-world testing of osimertinib drug levels using volumetric adsorptive microsampling in patients with NSCLC has not been conducted.

Several reports have described the bioanalysis of osimertinib and its metabolites in the plasma of patients with NSCLC13,30–33; however, the quantification of these drug metabolites using a DBS microsampling platform has only recently emerged. This article describes the development and validation of an ultraperformance liquid chromatographic tandem mass spectrometric method (UHPLC-MS/MS) for the quantitative analysis of osimertinib and its active metabolites, AZ5104 and AZ7550, in DBS samples collected using a hemaPEN microsampling device and a matched analysis of human plasma.

MATERIALS AND METHODS

Chemical and Materials

Osimertinib (purity 98.0%), AZ5104 (purity ≥95.0%), [13C2H3]-AZ7550 (purity ≥95.0%), and [13C2H3]-osimertinib (I.S.; purity ≥98%) were purchased from AlsaChim Co. Ltd (France). Acetonitrile (ACN; HPLC grade) and formic acid (A.R. grade) were purchased from Thermo Fisher Scientific Inc (Waltham, MA). Ammonium formate was purchased from Sigma Aldrich (St Louis, MO). Ultrapure water was obtained using Milli-Q Reagent Water System (Millipore, Bedford, MA). The hemaPEN was supplied by Trajan Medical & Scientific (Ringwood, VIC, Australia).

Instrumentation

The LC-MS system comprised a Shimadzu Nexera UHPLC with a dual pump, a vacuum solvent degasser, a controlled temperature autosampler maintained at 4°C, and an 8050 triple quadrupole MS/MS detector (Shimadzu, Kyoto, Japan) with a turbo-ion ESI source operating in the positive-ion multiple reaction monitoring (MRM) mode. Analytical separation was performed on an ACE PFP C18 column (2.1 × 100 mm, 1.7 µm; Hichrom, Reading, United Kingdom), thermostatically controlled at 50°C with a total run time of 6 minutes. The mobile phase consisted of 10 mmol/L ammonium formate in water (pH 4.3), 0.1% (v/v) formic acid (solution A), and ACN (solution B). The total flow rate was maintained at 0.3 mL/min. The gradient program is shown in Supplemental Digital Content 1 (see Table, http://links.lww.com/TDM/A695). The autosampler temperature was set to 4°C with an injection volume of 1 μL. The analytes were detected at an ion-spray voltage of 4.0 kV, interface temperature of 300°C, heating gas flow of 10 L/min, DL temperature of 250°C, and nebulizing gas flow rate of 2 L/min. The MRM transitions were m/z = 500.2→72.1, m/z = 486.3→72.1, m/z = 490.2→433.1, and m/z = 504.2→72.1 for osimertinib, AZ5104, [13C2H3]-AZ7550, and I.S, respectively. Because AZ7550 could not be commercially sourced at the time of this study, [13C2H3]-AZ7550 was used as the standard compound for this metabolite. The MS/MS parameters of each analyte are listed in Supplemental Digital Content 2 (see Table, http://links.lww.com/TDM/A695).

Preparation of Stock Solutions, Calibration Standards, and Quality Control Samples

Osimertinib, AZ5104, [13C2H3]-AZ7550, and I.S. were dissolved in ACN to obtain stock solutions with a final concentration of 1 mg/mL. Stock solutions were diluted using ACN/water (1:1, v/v) to prepare the working solution concentration of 10 mcg/mL. Calibration standard samples were prepared by spiking the corresponding working solutions with ethylenediaminetetraacetic acid (EDTA) whole blood (for DBS analysis) and EDTA plasma. The final calibration standard concentrations for osimertinib and its metabolites were 729, 243, 81, 27, 9, 3, and 1 ng/mL. Similarly, lower limit of quantification [LLOQ] and QC samples were prepared by diluting the working solutions with plasma at 4 concentrations: 250 ng/mL (high-quality control [HQC]), 30 ng/mL (medium-quality control [MQC]), 3 ng/mL (low-quality control [LQC]), and 1 ng/mL (LLOQ). All stock and working solutions were stored at −80°C until further analysis. All hemaPEN DBS calibration and QC standards were prepared by touching the tip of all 4 capillaries to the surface of a drop of blood, which was placed on a hydrophobic surface to simulate a finger-prick droplet. The device was held in a tilted position for 10 seconds to fill the capillaries, then inverted and closed with the cap to trigger the emptying of the whole capillary volume onto the precut DBS disks inside the hemaPEN. DBS samples were left to dry at room temperature (RT) for 1 hour inside the hemaPEN device before use. In the case of delayed analysis, the hemaPEN were stored at −80°C until use. For analysis, the DBS was retrieved from the device and transferred into a microcentrifuge tube.

Plasma Sample Preparation and Extraction

A total of 20 µL EDTA plasma was mixed with 50 µL I.S. (30 ng/mL). To this, 150 µL water and 650 µL ACN were added to precipitate the protein. After thoroughly vortexing for 5 minutes, the mixture was centrifuged at 15,000g for 10 minutes. One microliter of supernatant was injected into the UHPLC-MS/MS system.

DBS Sample Preparation and Extraction

A single DBS paper spot (2.74 μL) from the hemaPEN device was transferred into a 2-mL safe-lock vial containing 350 µL of ACN and 50 µL I.S. (30 ng/mL). After vortexing for 10 seconds, the mixtures were centrifuged at 15,000g for 5 minutes and then gently vortexed at RT for 1 hour. One microliter of supernatant was injected into the UHPLC-MS/MS system.

Method Validation

The method validation criteria included linearity, LLOQ, precision and accuracy, matrix effect, extraction recovery, stability, and carryover as described in the FDA and EMA bioanalytical validation guidelines. In addition, the International Association for Therapeutic Drug Monitoring and Clinical Toxicology (IATDMCT) DBS guidelines for assessing the hematocrit effect was addressed.34

The calibration curves contained 7 points based on the internal standard calibration and theoretical concentration, and the degree of fitness described by the correlation coefficient (r2) was >0.99. These criteria are described in Section 2.3.

QC samples were analyzed to assess the precision and accuracy of the assay. Six replicate analyses were conducted for each run at each QC concentration. The precision and accuracy were denoted by the relative SD (RSD) and relative error, respectively. The interrun and intrarun precision of LQC, MQC, and HQC samples were within ±15% of the corresponding concentration and ˂20% RSD for LLOQ.

The matrix effects of osimertinib, AZ5104, and AZ7550 were investigated using the matrix factor, which is the peak area ratio of the analyte/IS with 3 QC concentrations in the presence of matrix ions (plasma and DBS) to those in the absence of the matrix at equivalent concentrations. The RSD of the matrix effect was ≤15%. Recovery was assessed by comparing the peak areas obtained from the extracted QC samples with those in the mobile phase at the same concentrations and was expressed as a percentage.

Stability was tested under 4 different conditions: short-term stability, where QC samples were kept at RT for 6 hours and then analyzed; autosampler stability, where the processed QC samples were placed in an autosampler (4°C) for 24 hours and then evaluated; freeze–thaw stability, where QC samples were stored at RT for 8 hours, subjected to 3 freeze–thaw cycles (thawing at RT for 2 hours and freezing again at −80°C for at least 12 hours), and then analyzed; and long-term stability, where QC samples were analyzed after being stored at −80°C for 30 days.

The hematocrit effect was assessed at 4 levels (30%, 40%, 50%, and 60%), as they represented the range of the hematocrit in patient samples. The LQC, MQC, and HQC were measured at each hematocrit level. The relative errors of accuracy and the relative SD were determined. Statistical analysis was conducted using a 1-way analysis of variance.

Application of the Method to Clinical Samples

The Northern Sydney Local Health District Human Research Ethics Committee approved this study (2021/ETH00492). Written informed consent was obtained from all participants.

A single steady-state plasma and DBS trough sample was collected before the once-daily administration of 80 mg of osimertinib in 15 patients with NSCLC. To ensure steady-state sampling, patients were required to take osimertinib for 2 weeks before sample collection. DBS samples were obtained by lancet puncturing the participant's fingertip and collecting using a hemaPEN device. The sample was dried in the hemaPEN device at ambient temperature for 1 hour and then stored at −80°C.

Blood samples were obtained from the same patient through venipuncture within 10 minutes of DBS sample collection. Venous blood was collected in EDTA blood collection tubes, which were centrifuged at 2000g for 10 minutes to obtain the plasma, which was aliquoted and stored at −80°C. DBS and plasma concentrations were compared using Bland–Altman analysis.

RESULTS

Chromatography and LC-MS/MS Method

The drug analytes were assessed using positive ionization LC/MS in MRM mode. The precursor and product ions were determined by directly injecting a standard solution of osimertinib, AZ5104, [13C2H3]-AZ7550, and I.S. into the MS (Fig. 1). The source/gas and compound parameters were systematically optimized to achieve the optimal sensitivity (see Table, Supplemental Digital Content 2, http://links.lww.com/TDM/A695).

FIGURE 1.

FIGURE 1.

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Chemical structures, molecular weights, and product ion mass spectra of osimertinib and its metabolites, AZ5104 and AZ7550 (A). Product ion spectra (scan range 50–500 amu) of osimertinib (m/z = 500.3→72.0; B), AZ5104 (m/z = 486.2→72.0; C), and [13C2H3]-AZ7550 (m/z = 490.2→433.1; D).

The chromatographic conditions were assessed using commercially available columns and mobile phase gradient compositions. Compounds were separated using an ACE PFP C18 column (2.1 × 100 mm, 1.7 µm; Hichrom) with good peak symmetry and retention times, and LC/MS response was achieved following gradient elution with ammonium formate (10 mmol/L, pH 4.3) and ACN. The retention times for osimertinib, AZ5104, [13C2H3]-AZ7550, and I.S. were 3.8, 2.98, 3.5, and 3.8 minutes, respectively.

Optimization of Sample Preparation for Plasma and DBS

The extraction method, solvent composition, and volume were optimized to obtain the best extraction efficiency. Various extraction protocols are available for plasma and DBS35–39 with protein precipitation using ACN or methanol, owing to the simplicity of the procedure and acceptable analyte recovery. Osimertinib and its metabolites showed higher recoveries with ACN than with either methanol or MTBE. To optimize the sample volume for plasma, 3 different sample volumes (20, 50, and 100 µL) were tested, and the peak area responses were compared. Each volume produced similar peak areas; therefore, to reduce any matrix effects from higher sample volumes, the lower volume of 20 µL was selected as the optimal sample volume (see Figure, Supplemental Digital Content 1, http://links.lww.com/TDM/A695).

Similarly, different solvent compositions were tested for DBS to determine the best extraction efficiencies for osimertinib and its metabolites. All analytes showed good efficiency, larger peak areas, and higher recoveries with ACN than with methanol, methanol:water, or MTBE (see Figure, Supplemental Digital Content 2, http://links.lww.com/TDM/A695).

Analytical Method Validation

Selectivity and Specificity

Results were linear (r2 ≥ 0.99) for osimertinib, AZ5104, and AZ7550 in human plasma and DBS at concentrations ranging from 1 to 729 ng/mL. No significant chromatographic interferences were observed for osimertinib or I.S. at their defined retention times; however, occasional peak broadening was observed for the metabolites in the patient samples (Fig. 2). This could be due to the interactions with matrix components because cancer patients commonly take numerous medications independent of their therapeutic dosing. The peak areas for all compounds and I.S. in the blank plasma and DBS samples were <20% of the LLOQ response and <5% of the I.S., respectively, as required by the FDA, IATDMCT, and EMA guidelines. Typical chromatograms of blank human plasma and DBS; blank plasma and DBS samples mixed with osimertinib, AZ5104, AZ7550, and I.S. at LLOQ levels; and a patient steady-state sample (80 mg osimertinib) are shown in Figure 2. Chromatograms illustrating blank human plasma and DBS and blank plasma and MQC-level analyte-mixed DBS samples are shown in Supplemental Digital Content 3 (see Figure, http://links.lww.com/TDM/A695).

FIGURE 2.

FIGURE 2.

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Overlayed XIC chromatogram of (A) blank plasma and (B) blank DBS sample. (C, E, G, I) Plasma mixed with osimertinib, I.S., AZ5104, and [13C2H3]-AZ7550 at the LLOQ level (1 ng/mL). (D, F, H, J) DBS mixed with osimertinib, I.S., AZ5104, and [13C2H3]-AZ7550 at the LLOQ level (1 ng/mL). (K–N) Patient plasma samples with osimertinib, I.S., AZ5104, and AZ7550. (O–R) Patient DBS samples with osimertinib, I.S., AZ5104, and AZ7550. *Analyte elution at the desired RT.

LLOQ, Precision, and Accuracy

The intrabatch and interbatch precision and accuracy data for the determination of osimertinib, AZ5104, and [13C2H3]-AZ7550 at the LLOQ and 4 QC levels are summarized in Table 1. The average bias of the QC samples was <15% compared with the nominal concentration, and the RSD of each concentration level was <15%. The results demonstrated that the precision and accuracy were within acceptable limits and that the method was reliable and reproducible for the determination of osimertinib and its metabolites in both human plasma and DBS samples.

TABLE 1.

Intraday and Interday Accuracy and Precision of Quality Control Samples of Osimertinib, AZ5104, and [13C2H3]-AZ7550 (n = 6)

Plasma DBS
Analyte Nominal Concentration (ng/mL) Within-Run Precision (%) Between -Run Precision (%) Within-Run Accuracy (%) Between-Run Accuracy (%) Analyte Nominal Concentration (ng/mL) Within-Run Precision (%) Between -Run Precision (%) Within-Run Accuracy (%) Between-Run Accuracy (%)
Osimertinib 1 (LLOQ) 2.3 8.5 93.2 94.3 Osimertinib 1 (LLOQ) 10.3 4.3 96.7 90.2
3 (LQC) 3.6 1.6 98.9 99.7 3 (LQC) 7.5 1.0 96.1 99.4
30 (MQC) 1.0 0.7 97.6 99.9 30 (MQC) 4.6 0.1 99.4 98.9
270 (HQC) 0.2 0.4 99.3 99.8 270 (HQC) 0.5 0.2 99.6 99.3
AZ5104 1 (LLOQ) 10.0 9.8 98.4 97.1 AZ5104 1 (LLOQ) 15.5 3.5 90.9 92.7
3 (LQC) 2.8 3.2 96.3 98.7 3 (LQC) 7.5 2.8 99.7 99.5
30 (MQC) 0.6 0.2 98.1 99.9 30 (MQC) 0.9 0.2 99.5 98.6
270 (HQC) 0.2 0.3 98.6 99.3 270 (HQC) 0.6 0.2 99.8 99.4
AZ7550 1 (LLOQ) 6.9 3.7 99.7 96.1 AZ7550 1 (LLOQ) 6.6 2.9 95.1 94.5
3 (LQC) 1.5 1.7 98.9 98.5 3 (LQC) 6.4 2.5 97.5 99.3
30 (MQC) 1.3 0.5 98.4 99.1 30 (MQC) 1.1 0.4 99.7 99.1
270 (HQC) 0.5 0.4 99.2 99.2 270 (HQC) 1.3 0.1 98.6 99.4
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Matrix Effect and Recovery

The peak areas of the 6 extracted samples were compared with the mean peak areas of the 6 pure solutions at the LLOQ, LQC, MQC, and HQC concentrations. As shown in Table 2, the RSD of the I.S.-normalized matrix factors of osimertinib, AZ5104, and [13C2H3]-AZ7550 in plasma at the 4 QC levels ranged from 1.5% to 9.1%. In DBS, the RSD was in the range of 1.7%–9.2%, which suggests that no endogenous substance could cause ion suppression. The overall mean extraction recovery of osimertinib, AZ5104, and [13C2H3]-AZ7550 in plasma were in the range of 92.72 ± 6.6% to 101.8 ± 3.18%, and in DBS, the recovery was in the range of 91.89 ± 4.10% to 103.8 ± 6.66%, indicating no significant loss during the extraction process. Overall, the results indicated that high recovery and no significant matrix effects affected the successful validation of the method for osimertinib and its metabolites in plasma and DBS.

TABLE 2.

Matrix Effect and Extraction Recovery of Osimertinib, AZ5104, and [13C2H3]-AZ7550 in Plasma and DBS (n = 6)

Plasma Matrix Effect Plasma Recovery DBS Matrix Effect DBS Recovery
Analytes Concentration (ng/mL) Mean ± SD (ng/mL) RSD (%) Mean ± SD (ng/mL) RSD (%) Analytes Concentration (ng/mL) Mean ± SD (ng/mL) RSD (%) Mean ± SD (ng/mL) RSD (%)
Osimertinib 1 (LLOQ) 103.4 ± 5.1 5.1 92.7 ± 6.6 6.5 Osimertinib 1 (LLOQ) 98.4 ± 3.1 3.1 92.9 ± 3.7 3.8
3 (LQC) 101.9 ± 3.9 3.9 101.8 ± 3.1 3.1 3 (LQC) 101.9 ± 4.9 4.8 97.9 ± 5.4 5.5
30 (MQC) 109.2 ± 8.7 8.6 101.2 ± 1.4 1.5 30 (MQC) 101.2 ± 4.7 4.6 95.2 ± 7.4 7.3
270 (HQC) 108.1 ± 7.4 7.4 95.5 ± 1.9 2.0 270 (HQC) 106.1 ± 5.4 5.5 98.9 ± 5.5 5.6
AZ5104 1 (LLOQ) 110.3 ± 3.8 3.9 98.4 ± 4.7 4.6 AZ5104 1 (LLOQ) 90.3 ± 3.8 3.9 91.8 ± 4.1 4.2
3 (LQC) 107.0 ± 4.9 4.9 101.4 ± 7.4 7.3 3 (LQC) 106.8 ± 4.4 4.5 100.9 ± 3.6 3.5
30 (MQC) 108.8 ± 9.4 9.5 92.1 ± 9.1 9.1 30 (MQC) 103.8 ± 3.4 3.5 96.6 ± 4.2 4.3
270 (HQC) 104.1 ± 8.9 8.8 97.1 ± 2.4 2.5 270 (HQC) 101.1 ± 1.9 1.8 101.2 ± 8.8 8.9
AZ7550 1 (LLOQ) 113.8 ± 3.2 3.3 99.9 ± 7.1 7.1 AZ7550 1 (LLOQ) 103.8 ± 5.2 5.3 91.4 ± 8.1 8.2
3 (LQC) 102.8 ± 4.1 4.2 101.3 ± 8.5 8.4 3 (LQC) 101.9 ± 3.1 3.2 103.8 ± 6.6 6.5
30 (MQC) 103.0 ± 1.7 1.7 96.7 ± 7.1 7.1 30 (MQC) 100.7 ± 4.7 4.7 95.4 ± 5.7 1.7
270 (HQC) 101.8 ± 2.8 2.9 100.3 ± 6.8 6.9 270 (HQC) 97.8 ± 3.8 3.9 103.1 ± 9.1 9.2
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Stability

Storage stability experiments for osimertinib and its metabolites were assessed in both plasma and DBS at 3 different temperatures, namely, RT, 4°C, and −80°C, over various periods of 2 hours to 30 days. The results showed no significant degradation of osimertinib and its metabolites at −80°C in both plasma and DBS for at least 1 month. All compounds were found to be stable at 4°C for at least 72 hours. Compounds were stable for up to 5 hours at RT in plasma; however, after 1 day of storage at RT, the concentration of osimertinib and its metabolites decreased by >30%, which agrees with the findings of Rood et al,30 where they observed rapid degradation of osimertinib at RT. This may be attributed to the formation of irreversible Michael adducts with nucleophiles such as plasma albumin, which contribute to its instability in the plasma. By contrast, osimertinib and its metabolites were stable for up to 10 days at RT when stored as DBS (see Table, Supplemental Digital Content 3, http://links.lww.com/TDM/A695 and see Figure, Supplemental Digital Content 4, http://links.lww.com/TDM/A695).

Short-term storage in the autosampler (4°C) demonstrated that all compounds were stable for at least 48 hours, which is in line with other studies.40 In addition, plasma-extracted and DBS-extracted samples remained stable after 3 freeze–thaw cycles (from −80 degrees to 4°C) with a measured concentration within ±15% of the nominal concentration (Table 3).

TABLE 3.

Autosampler, Freeze–Thaw (F/T), and Long-Term Stability Data of Osimertinib, AZ5104, and [13C2H3]-AZ7550 in Plasma and DBS (n = 6)

Plasma DBS
Analytes Stability Type Concentration (ng/mL) Mean RSD (%) RE (%) Analytes Stability Type Concentration (ng/mL) Mean RSD (%) RE (%)
Osimertinib Autosampler 1 (LLOQ) 1.04 12.05 7.21 Osimertinib Autosampler 1 (LLOQ) 1.07 10.36 7.15
3 (LQC) 2.96 7.41 −1.01 3 (LQC) 2.96 7.51 −1.11
30 (MQC) 29.32 4.61 −2.3 30 (MQC) 29.31 4.63 −2.7
270 (HQC) 279.56 0.77 0.58 270 (HQC) 271.88 0.58 0.69
3 F/T cycles 1 (LLOQ) 1.14 9.87 10.47 3 F/T cycles 1 (LLOQ) 1.06 9.99 8.95
3 (LQC) 3.02 7.01 0.07 3 (LQC) 3.01 7.08 −0.43
30 (MQC) 28.84 3.23 −3.85 30 (MQC) 29.34 4.58 −2.24
270 (HQC) 269.25 1.00 −0.27 270 (HQC) 271.92 0.59 0.24
Long-term (−80 degrees) for 30 d 1 (LLOQ) 1.15 9.44 15.47 Long-term (−80 degrees) for 30 d 1 (LLOQ) 1.1 9.89 10.68
3 (LQC) 2.91 9.25 −2.7 3 (LQC) 2.91 6.69 0.14
30 (MQC) 29.39 4.55 −2.02 30 (MQC) 30.08 4.53 −2.18
270 (HQC) 270.31 1.2 0.11 270 (HQC) 269.97 0.59 0.71
AZ5104 Autosampler 1 (LLOQ) 1.01 9.79 1.80 AZ5104 Autosampler 1 (LLOQ) 1.03 9.79 1.65
3 (LQC) 2.97 2.29 0.84 3 (LQC) 2.99 2.29 −0.81
30 (MQC) 29.47 0.95 1.82 30 (MQC) 29.38 0.95 −1.7
270 (HQC) 272.01 0.5 0.73 270 (HQC) 272.01 0.50 0.63
3 F/T cycles 1 (LLOQ) 1.03 9.19 3.54 3 F/T cycles 1 (LLOQ) 1.05 9.33 5.05
3 (LQC) 2.99 2.36 0.26 3 (LQC) 3.07 2.58 0.24
30 (MQC) 29.47 0.94 1.76 30 (MQC) 28.84 2.44 −3.85
270 (HQC) 272.02 0.49 0.74 270 (HQC) 272.02 0.49 0.74
Long-term (−80 degrees) for 30 d 1 (LLOQ) 1.05 8.90 5.27 Long-term (−80 degrees) for 30 d 1 (LLOQ) 1.1 8.90 10.06
3 (LQC) 3.01 2.52 0.31 3 (LQC) 3.05 2.54 1.91
30 (MQC) 29.48 0.92 1.71 30 (MQC) 28.95 2.08 −3.48
270 (HQC) 272.02 0.48 0.75 270 (HQC) 270.24 0.57 0.08
AZ7550 Autosampler 1 (LLOQ) 1.01 6.61 0.25 AZ7550 Autosampler 1 (LLOQ) 1.01 6.22 1.55
3 (LQC) 2.96 6.42 1.01 3 (LQC) 2.96 6.31 −0.61
30 (MQC) 29.55 1.18 1.49 30 (MQC) 29.23 1.79 −1.7
270 (HQC) 272.16 1.31 0.80 270 (HQC) 272.17 1.30 0.61
3 F/T cycles 1 (LLOQ) 1.01 5.27 1.96 3 F/T cycles 1 (LLOQ) 1.03 5.12 5.03
3 (LQC) 2.98 6.53 0.43 3 (LQC) 3.02 6.44 0.26
30 (MQC) 29.56 1.16 1.44 30 (MQC) 29.08 1.71 −3.55
270 (HQC) 272.17 1.31 0.81 270 (HQC) 272.16 1.31 0.71
Long-term (−80 degrees) for 30 d 1 (LLOQ) 1.06 6.59 8.79 Long-term (−80 degrees) for 30 d 1 (LLOQ) 1.12 13.12 12.02
3 (LQC) 3.04 6.67 0.13 3 (LQC) 2.95 7.18 −1.94
30 (MQC) 29.16 1.69 2.79 30 (MQC) 29.21 1.90 −2.61
270 (HQC) 269.03 1.56 0.35 270 (HQC) 268.91 1.05 −0.41
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Effect of the Hematocrit

The hematocrit levels are influenced by factors such as individual and disease states. Patient samples had a hematocrit range of 33%–59%. The relative errors of osimertinib, AZ5140, and [13C2H3]-AZ5770 measured in DBS at 3 QC levels and 4 hematocrit levels (30%–60%) were assessed (Table 4), and no significant differences in accuracy were observed for hematocrit when tested using a 1-way analysis of variance.

TABLE 4.

Effects of Hematocrit on Osimertinib and Its Metabolites in DBS Samples (n = 5)

Hematocrit Levels (Osimertinib) Nominal Concentration (ng/mL) RE (%) RSD (%)
30% 3 (LQC) 96.5 13.2
30 (MQC) 101.5 4.8
220 (HQC) 96.5 5.1
40% 3 (LQC) 95.3 6.3
30 (MQC) 97.1 10.1
220 (HQC) 94.1 11.3
50% 3 (LQC) 98.7 9.4
30 (MQC) 94.9 10.5
220 (HQC) 103.6 7.5
60% 3 (LQC) 101.9 3.4
30 (MQC) 98.7 8.3
220 (HQC) 94.5 6.5
Hematocrit levels (AZ5104) Nominal concentration (ng/mL) RE (%) RSD (%)
30% 3 (LQC) 91.6 6.2
30 (MQC) 98.3 5.2
220 (HQC) 96.4 4.7
40% 3 (LQC) 90.7 6.1
30 (MQC) 92.1 7.9
220 (HQC) 91.1 9.0
50% 3 (LQC) 94.7 7.4
30 (MQC) 95.9 9.3
220 (HQC) 90.7 2.8
60% 3 (LQC) 95.9 7.4
30 (MQC) 96.2 6.3
220 (HQC) 96.5 1.8
Hematocrit levels ([13C2H3]-AZ7550) Nominal concentration (ng/mL) RE (%) RSD (%)
30% 3 (LQC) 90.6 6.2
30 (MQC) 98.1 5.7
220 (HQC) 90.3 3.3
40% 3 (LQC) 92.7 4.3
30 (MQC) 97.1 7.1
220 (HQC) 90.3 2.4
50% 3 (LQC) 92.7 5.4
30 (MQC) 94.7 9.5
220 (HQC) 92.7 5.5
60% 3 (LQC) 95.2 6.1
30 (MQC) 94.7 5.3
220 (HQC) 93.9 2.6
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Correlation Between Plasma and Finger-Prick DBS Values

The developed and validated method was used to measure the concentrations of osimertinib, AZ5104, and AZ7550 in the plasma and microsampled DBS from 15 patients with NSCLC (see Table, Supplemental Digital Content 4, http://links.lww.com/TDM/A695). Figure 3 shows the steady-state drug levels in patients with NSCLC treated with an oncologist-recommended starting dosage of 80 mg after at least 14 days of treatment. The median plasma concentrations of osimertinib, AZ5104, and AZ7550 were 205 ng/mL (range 166.6–285.8 ng/mL), 20 ng/mL (13.2–23.9 ng/mL), and 18 ng/mL (13.2–23.2 ng/mL), respectively. In DBS, median concentrations of osimertinib, AZ5104, and AZ7550 were 203 ng/mL (range, 183.4–256.6 ng/mL), 19 ng/mL (14.9–23.6 ng/mL), and 18 ng/mL (14.7–20.6 ng/mL), respectively. These steady-state concentrations were within the ranges previously reported for 80-mg dosing in patients with NSCLC.41–45 The Bland–Altman mean difference plot had high concordance between plasma and finger-prick DBS measurements (Fig. 4). For osimertinib, AZ5104, and AZ7550, a mean concentration difference of 0.4%, −3.5%, and −3.4% between plasma and hemaPEN DBS was observed, respectively, indicating no significant difference for measuring osimertinib with a minor plasma bias for both metabolite concentrations. The strong agreement between the microsampled DBS and plasma concentrations is consistent with the reported osimertinib blood-to-plasma drug ratio of 1 observed in human participants13 and dogs.46 Similar blood-to-plasma ratios have been reported for other TKIs.47,48

FIGURE 3.

FIGURE 3.

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Steady-state plasma (A) and DBS (B) concentration of osimertinib (red circles), AZ5104 (blue squares), and AZ7550 (gold triangles) from the patients with NSCLC who were prescribed 80 mg daily dose (n = 15, average shown by line).

FIGURE 4.

FIGURE 4.

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Bland–Altman analysis of (A) osimertinib, (B) AZ5104, and (C) AZ7550 comparing the DBS and plasma concentrations from 15 patients with NSCLC. Dotted lines represent limit of agreement (±1.96 SD of the mean difference).

DISCUSSION

In this study, the development and validation of an UHPLC-MS/MS assay to measure osimertinib and its 2 metabolites, AZ5104 and AZ7550, from EDTA plasma and DBS of patients with NSCLC was described. For the first time, the applicability of whole-blood microsampling for measuring these compounds was demonstrated in DBS at clinically relevant concentrations. Investigation of the hematocrit effect revealed no impact on the measurement accuracy of any of the assayed compounds. This comparison of osimertinib measured in the plasma and whole blood showed an equivalence consistent with the reported blood-to-plasma ratio of 1.13 Therefore, this study supports the future use of microsampling and hemaPEN DBS analysis for TDM of osimertinib, which may address the issue of toxic exposures often experienced by patients with cancer.15–17

In the stability study, osimertinib and its metabolites were stable in the DBS for at least 10 days at RT. The long-term stability of the compound at RT is an important advantage that allows blood samples to be obtained from various facilities, including those at more remote locations that require shipping to a more equipped laboratory for analysis. As DBS microsampling requires no specialized postsampling equipment (eg, a centrifuge or refrigerator), this method facilitates self-collection by the patient, which may improve equity in patient management and eliminate the need for venipuncture.

The concentration of AZ7550 measured in patient samples was determined using the 13C-labeled standard compound [13C2H3]-AZ7550, as the unlabeled compound was unavailable at the time of this study. The basis for this approach is that [13C2H3]-AZ7550 is an isotopologue of AZ7550 expected to display signal responsiveness equivalent to that of AZ7550, and we confirmed the assay specificity for AZ7550 detection in patient samples (see Figure, Supplemental Digital Content 5, http://links.lww.com/TDM/A695). Any error in determining the absolute concentration of AZ7550 in the patient specimens using this quantitation strategy was assumed to be negligible.

CONCLUSIONS

A sensitive and selective UHPLC-MS/MS method was developed and validated for the simultaneous analysis of osimertinib, AZ5104, and AZ7550 in human EDTA plasma and DBS using hemaPEN microsampling technology. The microsampling DBS approach provided accurate quantities of these 3 analytes in blood, with a hematocrit range of 30%–60%. Because all relevant acceptance criteria required by the FDA and EMA bioanalytical guidelines and IATDMCT DBS guidelines were met, it can be concluded that the hemaPEN allows the accurate and precise quantitation of osimertinib in whole blood. This could be useful for TDM to manage drug-related toxicity and for other drug pharmacokinetics purposes.

Supplementary Material

tdm-46-332-s001.docx (3.3MB, docx)
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ACKNOWLEDGMENTS

The authors thank Alex Anderson and Andrew Gooley from Trajan Scientific for the gracious gift of hemaPEN. The clinical aspects of this project were supported by J Wei, L Gray, S Miller, and B Kirwin. This research was facilitated by access to Sydney Mass Spectrometry, a core research facility at the University of Sydney.

Footnotes

N. Pavlakis Astra Zeneca-Advisory Board Honoraria. M. Itchins Astra Zeneca- Honoraria. S. J. Clarke Astra Zeneca–Expert testimony, Advisory Board honoraria. The authors declare no conflict of interest.

A. Yuile, S. J. Clarke, and M. P. Molloy developed the concept. Method development, clinical validation, and data analysis were conducted by B. Venkatesh, M. J. McKay, and M. P. Molloy. A. Yuile, S. Narayanan, H. Wheeler, M. Itchins, N. Pavlakis, and S. J. Clarke provided resources, specimens, and clinical data. B. Venkatesh wrote the manuscript. AY, M. J. McKay, S. J. Clarke, and M. P. Molloy reviewed and edited the manuscript. A. Yuile and M. P. Molloy supervised the study. All the authors have read and approved the final version of the manuscript.

Supplemental digital content is available for this article. Direct URL citations appear in the printed text and are provided in the HTML and PDF versions of this article on the journal's Web site (www.drug-monitoring.com).

Contributor Information

Bharat Venkatesh, Email: bharat.venkatesh@sydney.edu.au.

Alex Yuile, Email: alexyuile92@gmail.com.

Matthew J. McKay, Email: matthew.mckay@sydney.edu.au.

Helen Wheeler, Email: helen.wheeler@health.nsw.gov.au.

Malinda Itchins, Email: malinda.itchins@sydney.edu.au.

Nick Pavlakis, Email: nick.pavlakis@sydney.edu.au.

Stephen J. Clarke, Email: stephen.clarke@sydney.edu.au.

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