Development and Validation of a Rapid LC-MS/MS Method for Carfilzomib Quantification in Plasma from Multiple Myeloma Patients

Summary

This study aimed to develop and validate a rapid, sensitive liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for the quantification of carfilzomib in human plasma. The fully validated method supports therapeutic drug monitoring in patients with multiple myeloma, thereby enabling personalized dose optimization.

Abstract

Carfilzomib, a second-generation proteasome inhibitor, requires therapeutic drug monitoring (TDM). This study aimed to develop and validate a sensitive and robust liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for the quantification of carfilzomib in human plasma. Plasma samples were processed using protein precipitation with acetonitrile (ACN). Chromatographic separation was achieved on a C₁₈ column (2.1 mm × 100 mm, 3.5 µm) maintained at 40 °C, with a mobile phase composed of ACN and 10 mmol/L ammonium acetate in water (80:20, v/v) delivered at a flow rate of 0.35 mL/min. The method demonstrated excellent linearity over a concentration range of 2.00-1000.00 ng/mL. In addition, high intra-day and inter-day precision and accuracy were observed, with recovery rates for carfilzomib ranging from 84.1%-93.0%. This validated LC-MS/MS method enables accurate, efficient, and sensitive determination of carfilzomib concentrations in plasma from patients with multiple myeloma, thereby supporting the optimization of carfilzomib-based therapy.

Introduction

Multiple myeloma (MM) is a hematological malignancy characterized by the clonal proliferation of plasma cells in the bone marrow¹. It represents the second most common hematological malignancy globally², accounting for approximately 10% of all hematological cancers³. Over the years, advancements in drug therapy, including the introduction of proteasome inhibitors, immunomodulatory drugs, and monoclonal antibodies, have led to improved therapeutic outcomes. However, a significant proportion (20%-30%) of MM patients continue to experience challenges such as short progression-free survival (PFS)

Protocol

This research protocol was approved by the Ethics Committee of Second Hospital of Naval Medical University (Shanghai Changzheng Hospital). All the patients enrolled in the study signed the informed consent.

1. LC-MS/MS instrumentation

1. Use an ultra-high-performance liquid chromatography (UHPLC) system coupled to a triple quadrupole mass spectrometer equipped with an electrospray ionization (ESI) source to perform method development and sample analysis.
2. Configure the UHPLC system to include an online degasser, a binary pump, an autosampler, and a column oven.
3. Conduct data acquisition and proces....

Results

LC-MS/MS optimization

To optimize the detection of carfilzomib, the response values of the analyte were compared under both positive and negative ionization modes. The negative ionization mode yielded a higher response for carfilzomib than the positive ionization mode and was therefore selected for subsequent analysis. We compared the performance of C₈, C₁₈, and T3-C₁₈ columns to select the optimal one. The C₁₈ column provided the be.......

Discussion

In contrast to traditional HPLC-UV and ELISA methods, LC-MS/MS provides exceptional selectivity, sensitivity, and speed, effectively addressing the limitations of its predecessors. These advantages solidify its role as the gold-standard technique for drug analysis in complex biomatrices, particularly for TDM and pharmacokinetic investigations¹⁷. We have developed and validated an LC-MS/MS method for quantifying carfilzomib in human plasma. A key practical advantage of this method is its simpl.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Acetonitrile (ACN)	Merck Company (Darmstadt, Germany)	34888	LC-MS grade
Ammonium acetate	Merck Company (Darmstadt, Germany)	9689	Analysis purity: ≥ 99.0%
Analysis balance	Sartorius (Germany))		Precision: 0.01 mg
Analytical column	Agilent Technologies (USA)		Agilent ZORBAX SB-C18, 2.1 × 100 mm, 3.5 µm
Carfilzomib	Shanghai yuanye biotechnology (Shanghai, China)	868540-17-4	Analyze the standard sample,purity > 99%
Carfilzomib-d8	Shanghai yuanye biotechnology (Shanghai, China)		Analyze the standard sample, purity > 98%
Data acquisition software	Agilent Technologies (USA)		Agilent Masshunter data processing software (version 6.0)
Data analysis software	Agilent Technologies (USA)		Agilent MassHunter quantitative analysis software (version 10.1)
Distilled water	Shenzhen Watsons Distilled Water Co., Ltd (Shenzhen, China)	LC-MS grade
HPLC Vials & Inserts	Shanghai Titan Technology Co., Ltd. ((Shanghai, China))		-
Liquid chromatography system	Agilent Technologies (USA)		Agilent 1200 series includes binary pumps, online degasser, automatic sampler, and column temperature box.
Methanol (MeOH)	Merck Company (Darmstadt, Germany)	1.06035	LC-MS grade
Micropipettes	Eppendorf (Germany)		Multiple measurement ranges (e.g., 10 µL, 100 µL, 200 µL, 1000 µL)
Office & Statistical software	-		Microsoft Office 365; GraphPad Prism 9.5
Phosphoric acid	Merck Company (Darmstadt, Germany)	49685	Analysis purity, 85 wt.%
Refrigerated centrifuge	Eppendorf (Germany)		For samples centrifuged at 4°C
Triple quadrupole mass spectrometer	Agilent Technologies (USA)	G6475AA	Agilent 1200 series, equipped with electrospray ionization source (ESI)
Vortex mixer	Labnet (USA)	S0200	Adjustable speed