Quantitation of osimertinib, alectinib and lorlatinib in human cerebrospinal fluid by UPLC-MS/MS
Simon P de Leeuw1, Peter de Bruijn2, Stijn L W Koolen3
1Department of Medical Oncology, Erasmus MC Cancer Institute, Rotterdam, the Netherlands; Department of Pulmonary Medicine, Erasmus MC Cancer Institute, University Medical Center, Rotterdam, the Netherlands.
Abstract:
Overall survival in metastatic lung cancer has been dramatically improved with the use of small molecule kinase inhibitors (SMKIs). Quantification of SMKI in cerebrospinal fluid (CSF) can be used to assess penetration of these drugs into the central nervous system. This paper describes an ultra-performance liquid chromatography tandem mass spectrometry (UPLC-MS/MS) method for quantification of the SMKIs alectinib, lorlatinib and osimertinib in human CSF. Alectinib-d8 and dasatinib-d8 were used as internal standards. Aliquots with 25 µL CSF/30% albumin (9:1,v/v) were mixed with 100 µL internal standard solution consisting of 1 ng/mL dasatinib-d8 and alectinib-d8 in acetonitrile. The analytes were separated by an Acquity UPLC® HSS T3 column (2.1 ×150 mm, 1.8 µm), using gradient elution (ammonium formate pH 4.5, acetonitrile) with a flow rate of 0.400 mL/min. All calibration curves were linear for the concentration range from 2.50 to 250 ng/mL. Within-run and between-run precision varied from 0.72% to 11.7%, with accuracy ranging from 95.3% to 113.2%. For all compounds, a high degree of non-specific binding to the vacutainer was observed. This issue could be countered easily by a combination of pre-coating with BSA solution (30%) in phosphate buffer pH 4.2, and immediate sample mixture with BSA solution after collection. To test the clinical applicability, CSF was collected in seven unique patients using alectinib (n = 1), lorlatinib (n = 2), and osimertinib (n = 4). Measured CSF trough concentrations ranged between 3.37 and 116 ng/mL.
Insights
This study developed a UPLC-MS/MS method to measure small molecule kinase inhibitors (SMKIs) in cerebrospinal fluid (CSF). The method accurately quantifies alectinib, lorlatinib, and osimertinib, aiding CNS drug penetration assessment for metastatic lung cancer.
Area of Science:
- Analytical Chemistry
- Pharmacology
- Oncology
Background:
- Small molecule kinase inhibitors (SMKIs) have significantly improved survival in metastatic lung cancer.
- Assessing SMKI penetration into the central nervous system (CNS) via cerebrospinal fluid (CSF) is crucial for treatment efficacy.
- Accurate quantification methods for SMKIs in CSF are needed to evaluate CNS drug delivery.
Purpose of the Study:
- To develop and validate an ultra-performance liquid chromatography tandem mass spectrometry (UPLC-MS/MS) method for quantifying alectinib, lorlatinib, and osimertinib in human CSF.
- To assess the clinical applicability of the developed UPLC-MS/MS method by measuring SMKI concentrations in patient CSF samples.
Main Methods:
- Ultra-performance liquid chromatography tandem mass spectrometry (UPLC-MS/MS) was employed for quantification.
- Alectinib-d8 and dasatinib-d8 served as internal standards.
- Sample preparation involved mixing CSF with internal standard solution and acetonitrile; separation was achieved using a HSS T3 column with gradient elution.
Main Results:
- The UPLC-MS/MS method demonstrated linear calibration curves (2.50–250 ng/mL) with high precision (0.72%–11.7%) and accuracy (95.3%–113.2%).
- Non-specific binding to vacutainers was observed and mitigated by pre-coating with BSA solution and immediate sample mixing.
- Clinical testing in seven patients showed measured CSF trough concentrations ranging from 3.37 to 116 ng/mL for the studied SMKIs.
Conclusions:
- A robust and accurate UPLC-MS/MS method for quantifying alectinib, lorlatinib, and osimertinib in human CSF has been successfully developed.
- The method addresses challenges like non-specific binding, enabling reliable assessment of SMKI CNS penetration.
- This validated method supports clinical studies evaluating the efficacy of SMKIs in treating brain metastases in lung cancer patients.
