MSACL 2024 Abstract

Introduction: Dabrafenib and trametinib are targeted drugs used for the treatment of metastatic melanoma and other un-resectable solid tumors with BRAF V600 mutations. The drugs inhibit two protein kinases: the mutated B-Raf kinase (dabrafenib) and its downstream MEK1/2 kinase (trametinib). Although the targeted therapies are highly effective and administered orally, they can also exert significant toxicity, resulting in dose reduction and discontinuation. To maximize treatment efficacy while minimizing adverse events, clinicians should make informed treatment decisions based on the therapeutic drug levels of dabrafenib, its major metabolite, and trametinib.

Objectives: The primary objective of the study was to develop and evaluate mass spectrometry-based methods to quantify dabrafenib, hydroxy-dabrafenib (OH-dabrafenib), and trametinib in plasma to monitor drug concentrations in metastatic melanoma patients receiving targeted therapy of dabrafenib and trametinib.

Methods: We developed liquid chromatography (LC) and paper spray methods coupled to tandem mass spectrometry (MS/MS) for the quantification of dabrafenib, OH-dabrafenib, and trametinib. In the LC method, 100 µL of plasma was aliquoted into an amber plastic sample vial and 300 µL of methanol (containing Dabrafenib-D9 and Trametinib-[13C]6 internal standards [IS]) was added. Next, the samples were mixed at 2000 rpm (at 5°C) for 5 minutes, followed by 5-minute centrifugation at 10,000 x g (at 5°C). 100 µL of the supernatant were transferred into an amber 300 µL glass autosampler vial and mixed with 80 µL of water. The resulting samples (10 µL) were analyzed by LC-MS/MS using Thermo Scientific TSQ Altis triple quadrupole MS with heated electrospray ionization in positive mode. A Thermo Scientific Hypersil Gold aQ (2.1x50mm, 3um) column, connected to a Thermo Scientific Vanquish Horizon SII autosampler and LC pumps, was used to achieve chromatographic separation utilizing a linear gradient with a total run time of 5.2 minutes. Mobile phase A and B contained 0.1% formic acid in water and methanol, respectively. Compounds were identified by retention time, relative retention time to an isotopically labeled IS, and Q1/Q3 ion pair ratios.
For the paper spray ionization (via Thermo Scientific VeriSpray ion source) method, 100 µL of plasma was aliquoted into an amber plastic sample vial and 200 µL of methanol IS was added. After the same vortex and centrifugation steps, 6 µL of the supernatant was transferred onto a VeriSpray sample plate. The sample plates were air dried under dark conditions for 30 minutes. The resulting samples were analyzed with a VeriSpray ion source connected to the TSQ Altis MS. Samples were ionized directly into the MS system by first rewetting the samples with 20 µL of 0.1% formic acid in 1:1 acetonitrile: isopropanol and then adding 100 µL of the spray solvent (0.01% formic acid in 9:1 methanol: water). Both rewetting and spray solvents were added in 10 µL increments with time delays between each respective solvent addition. Chronographs were generated by applying 3800 volts to the spray paper from time point 0.1 to 1.1 minutes. Compounds were identified by Q1/Q3 ion pair ratios and relative abundance to the IS response.

Results: The LC method development proved challenging due to the lack of an isotopically labeled IS for OH-dabrafenib. When using Dabrafenib-D9 as it's IS, we observed inconsistency in the OH-dabrafenib analyte response relative to Dabrafenib-D9 at higher concentrations. When evaluating the matrix effects of patients’ EDTA plasma, we visually observed variable ion signal (via t-infusion signal suppression) at the retention time of dabrafenib-D9 that was not present at the OH-dabrafenib retention time. To mitigate these signal variations, we analyzed the extracted samples in triplicate injections and the final calculated result was averaged. The replicate injections significantly improved imprecision. Across the analytic measuring range, the total imprecision for dabrafenib and trametinib was 1.4-6.8% and 1.4-3.4%, respectively. However, for OH-dabrafenib the total imprecision was 4.0-13.7%. The final LC method resulted in a linear analytic measurement range of 10-3500, 10-2500, and 0.5-50 ng/mL for dabrafenib, OH-dabrafenib, and trametinib, respectively.
Conversely, the paper spray method only utilizes a single “injection” per sample and did not display differential suppression for the three analytes and two ISs. In addition, the approximately 2.5-minute cycle time (including the needed pauses during solvent addition) of the paper spray ionization method yielded a faster turnaround time for the entire set of samples. While the paper spray method resolved the suppression observed for OH-dabrafenib, the overall signal intensity for trametinib at the lower limit of quantitation (LLOQ, 0.5 ng/mL) was not enough to reproducibly distinguish it from a matrix blank’s signal. Therefore, the paper spray method resulted in a linear analytic measurement range of 10-3500, 10-2500, and 2.0-50 ng/mL for dabrafenib, OH-dabrafenib, and trametinib, respectively.

Conclusions: We developed both LC and paper-spray ionization methods for the detection and quantification of dabrafenib, OH-dabrafenib, and trametinib in EDTA plasma. While the LC method provides a more sensitive detection limit for trametinib, the paper spray method provides more reproducible results for OH-dabrafenib, without an isotopically labeled IS. Overall, the paper spray provided a faster analysis for all of the analytes. Based on the laboratory’s needs, the adoption of either analytical method could provide accurate results.