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)^4,5 adverse reactions (ADRs), drug resistance, and relapse⁶.

^{Bortezomib, the first-in-class proteasome inhibitor7}, has transformed the therapeutic landscape of MM. Despite its efficacy, a majority of patients ultimately develop relapsed or refractory disease, frequently accompanied by bortezomib resistance^8,9 and reduced rates of treatment response and survival^10,11. These challenges underscore the urgent need for innovative treatment approaches to improve outcomes in MM. Carfilzomib, a second-generation proteasome inhibitor approved by the FDA, provides a more stable pharmacokinetic profile, enabling sustained suppression of chymotrypsin-like activity and delivering potent antimyeloma effects. It is generally indicated for MM patients who have undergone at least two prior lines of therapy, including treatment with bortezomib and an immunomodulatory drug^1,11. Research has shown that carfilzomib achieves more robust proteasome inhibition in myeloma cells than bortezomib and possesses the ability to surmount bortezomib resistance^12,13.

The most widely used methods for quantifying drug concentrations in biological matrices include high-performance liquid chromatography-ultraviolet detection (HPLC-UV), enzyme-linked immunosorbent assay (ELISA), and liquid chromatography-tandem mass spectrometry (LC-MS/MS)¹⁴. Although HPLC-UV offers operational simplicity and low cost, its utility is constrained by relatively low sensitivity and selectivity. Consequently, accurate quantification of target analytes, particularly at low concentrations or within complex biological matrices, remains challenging¹⁵. ELISA demonstrates high sensitivity. However, its application faces several limitations, including a lengthy development process, reliance on high-quality antibodies, and susceptibility to cross-reactivity with structurally similar compounds in the sample. These factors collectively compromise the specificity of the assay¹⁶. In contrast, LC-MS/MS offers exceptional selectivity, superior sensitivity, and rapid analytical throughput. These advantages establish it as the gold-standard methodology for therapeutic drug monitoring (TDM) and pharmacokinetic investigations¹⁷. Carfilzomib has the characteristics of extensive plasma protein binding, poor in vivo stability and a narrow therapeutic index. The LC-MS/MS technology can perform rapid quantitative analysis of the target compound, effectively reduce the matrix effect, and provide a scientific basis for precise clinical drug administration.

The drug exposure level of carfilzomib is closely related to its therapeutic effect and ADRs^18,19. Carfilzomib has a dose-dependent property²⁰, and its exposure level increases positively with the dose in the body, which may directly affect its therapeutic effect. A study showed that the complete response (CR) rate of carfilzomib in the treatment of MM was 28%, the very good partial response (VGPR) rate was 73%, and the overall response rate (ORR) was 93%²¹. In addition, compared with the control group, the incidence of cardiotoxicity in patients treated with carfilzomib was significantly increased, while there was no significant change in peripheral neuropathy. Another study also mentioned the efficacy and safety of carfilzomib in the treatment of MM and confirmed that carfilzomib may increase the risk of cardiotoxicity while improving the therapeutic effect²². Therefore, the efficacy and ADRs of carfilzomib are related to the exposure level.To date, few validated LC-MS/MS methods have been reported for the quantification of carfilzomib in human plasma, particularly those meeting rigorous bioanalytical validation criteria (e.g., FDA guidelines). Existing studies primarily focus on animal models or other biological matrices^23,24, highlighting a gap in clinically applicable assays for TDM in MM patients.

Therefore, there is a pressing need to establish a rapid, precise, and sensitive assay for monitoring carfilzomib concentrations in MM patients to optimize its therapeutic utility and safety profile. In this work, we developed and validated a reliable LC-MS/MS method capable of quantifying carfilzomib in human plasma within 3 min, demonstrating excellent sensitivity and reproducibility. Developed for TDM of carfilzomib in MM, this method supports dose optimization and exposure-response evaluation. Users must be aware of the drug's instability in plasma; accurate quantification necessitates sample processing within 4 h of blood draw.

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 processing using the instrument manufacturer's data acquisition software.

2. Liquid chromatographic conditions

1. Achieve chromatographic separation using a ZORBAX-SB C₁₈ column (2.1 mm × 100 mm, 3.5 µm).
2. Maintain the column temperature at 40 °C.
3. Conduct isocratic elution with a mobile phase comprising 20% 10 mmol/L ammonium acetate in water and 80% ACN.
4. Set the flow rate to 0.35 mL/min.
5. Ensure symmetrical peak shapes and suitable retention times for both the analyte and the internal standard (IS) under these conditions.
   NOTE: Carfilzomib-d8 was selected as the IS due to its structural similarity to carfilzomib, which ensures comparable extraction recovery, chromatographic behavior, and ionization efficiency.
6. Set the total run time to 3.0 min.

3. Mass spectrometry conditions

1. Operate the mass spectrometer in negative ESI mode.
2. Set the capillary voltage to -6000 V.
3. Employ nitrogen as both the nebulizing and drying gas.
4. Use high-purity nitrogen as the collision gas at a pressure of 0.2 MPa.
5. Maintain the nebulizer pressure at 20 psi.
6. Heat the drying gas to 400 °C and deliver it at a flow rate of 10 L/min.
7. Set the sheath gas temperature to 350 °C, with a flow rate of 12 L/min.
8. Acquire data in multiple reaction monitoring (MRM) mode.
9. Acquire product ion spectra of carfilzomib (Figure 1A) and the IS (Figure 1B) to illustrate their fragmentation patterns.
10. Use the MS2 Scan mode to identify the parent ion.
11. Use the MS2 SIM mode to optimize the Fragmentor voltage.
12. Use the Product Ion mode to determine the optimal Collision Energy (CE).
13. Detail the optimized mass spectrometry parameters for carfilzomib and the IS (Table 1).

4. Calibration and quality control samples

1. Prepare a stock solution of carfilzomib (1.0 mg/mL) by accurately weighing 2.0 mg of carfilzomib into a 2 mL volumetric flask and dissolving it in 2 mL of methanol (MeOH).
   CAUTION:MeOH is flammable and toxic. When operating, it should be done in a fume hood to prevent inhalation of vapors or skin contact. Use appropriate personal protective equipment, including gloves and eye/face protection.
2. Store the stock solution at -80 °C.
3. Generate working solutions through serial dilution of the stock solution with 10% MeOH to achieve concentrations of 100.0 µg/mL, 10.0 µg/mL, and 1.0 µg/mL.
4. Establish calibration standards by spiking blank plasma with the working solutions.
5. Dilute the spiked plasma with blank plasma at a ratio of 1:9 (v/v) to obtain final concentrations of 2.0, 5.0, 50.0, 200.0, 500.0, 750.0, and 1000.0 ng/mL.
6. Independently prepare quality control (QC) samples using the same method at concentrations of 5.0 ng/mL (low), 200.0 ng/mL (medium), and 750.0 ng/mL (high).

5. Sample preparation

1. Pretreat plasma samples (100 µL) by adding 20 µL of a 4% phosphoric acid solution.
   CAUTION: Phosphoric acid is corrosive. Wear gloves, safety goggles, and a lab coat to prevent skin and eye contact. In case of contact, rinse immediately with copious amounts of water.
2. Vortex-mixing the sample for 2 min at maximum speed using a standard laboratory vortex mixer.
3. Add 200 µL of ACN, ensuring it contains an IS solution at a concentration of 200.0 ng/mL, to the mixture²⁵.
   CAUTION: ACN is flammable, volatile and toxic. All operations must be carried out in a fume hood and kept away from heat sources and flames. Use appropriate personal protective equipment, including gloves and eye/face protection.
4. Vortex the combined mixture again.
5. Centrifuge the mixture at 12,500 ×g for 10 min at 4 °C²⁵.
6. Collect 100 µL of the supernatant.
7. Combine the 100 µL supernatant with 100 µL of water.
8. Transfer the resulting solution to an HPLC vial equipped with a glass insert.
9. Inject 10 µL of the final solution into the LC-MS/MS system for analysis.

6. Specificity assessment

1. Assess specificity by comparing the responses of carfilzomib and the IS in six different batches of blank plasma, IS-spiked plasma, the lower limit of quantification (LLOQ) sample, and authentic plasma samples.
2. Consider interference acceptable if the response is less than 15% of the LLOQ for the analyte and less than 5% of the IS response.

7. Linearity evaluation

1. Evaluate linearity by analyzing three sets of calibration standards on different days (at least two days).
2. Use a weighted linear least-squares regression model to generate calibration curves based on at least six calibration standards.
3. Define the lowest concentration standard as the LLOQ.
4. Require back-calculated concentrations for each standard to be within ± 15% of their nominal values, with a tolerance of ± 20% for the LLOQ.

8. Precision and accuracy determination

1. Determine intra-day and inter-day precision and accuracy by analyzing five replicates of QC samples at three concentration levels and the LLOQ sample on at least two different days.
2. Express precision as the relative standard deviation (RSD%), requiring it to be ≤ 15% for QC samples and ≤ 20% for the LLOQ.
3. Express accuracy as the relative error (RE%), requiring it to be within ± 15% for QC samples and ± 20% for the LLOQ.

9. Recovery and matrix effect determination

1. Determine recovery by comparing the peak area of carfilzomib spiked into plasma before extraction with the peak area of carfilzomib spiked into the extracted matrix at the same concentration.
2. Evaluate the matrix effect by comparing the peak area of carfilzomib spiked into the extracted matrix with the peak area of carfilzomib in a neat solution at the same concentration.
3. Indicate a consistent impact of the blank plasma matrix on the analyte signal if the RSD% for the matrix effect factor is less than 15%.

10. Carryover assessment

1. Assess carryover by injecting a blank sample immediately following the highest calibration standard to detect any residual analyte signal from the preceding injection.
2. Consider carryover acceptable if the response of carfilzomib in the blank sample is less than 20% of the LLOQ and less than 5% of the IS response.

11. Dilution effect evaluation

1. Evaluate the dilution effect by diluting spiked plasma samples with concentrations exceeding the upper limit of quantification (ULOQ) with blank plasma to bring the concentration within the calibration range.
2. Compare the measured concentration to the nominal concentration.

12. Stability assessment

1. Assess stability by analyzing QC samples at three concentration levels under different storage conditions.
2. Store QC samples at room temperature and analyze after 6 h.
3. Store processed samples in the autosampler and analyze at 0, 2, 4, and 6 h.
4. Subject QC samples to three freeze-thaw cycles by alternating storage at −20 °C and room temperature, then analyze.
5. Store QC samples at −80 °C for two months, then analyze.
6. Compare measured concentrations with nominal values and confirm that deviations are within ±15% (RE%).

13. Patient enrollment and sample collection

1. Obtain written informed consent from all patients.
2. Enroll patients with MM and treat them with carfilzomib.
3. Collect blood samples immediately after drug administration (IV infusion over 30 min).
4. Obtain plasma samples by centrifuging blood samples collected in EDTA-3K tubes at 4500 ×g for 10 min at 4 °C.

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