Development and Standardization of an Ex Vivo Micromethod for Intracellular Quantification of Vincristine in Primary ALL Cells by LC-MS/MS

Summary

This protocol aimed to establish and optimize an LC-MS/MS method for the relative quantification of vincristine accumulation in primary pediatric ALL cells.

Abstract

Quantifying intracellular vincristine (VCR) in primary pediatric acute lymphoblastic leukemia (ALL) cells is essential for understanding sample-specific differences in drug uptake and for supporting experimental studies using scarce patient material. Here, we present a streamlined LC-MS/MS micromethod specifically optimized for primary ALL cells obtained through patient-derived xenografts (PDX), addressing key challenges such as limited cell availability, small cell size, and the need for reproducible recovery after multiple washing steps. Time-course assays ranging from 1 to 5 h demonstrated that a 3 h incubation period yields the highest and most consistent intracellular VCR levels, with lower variability across replicates. Method optimization also established that using 2.5 × 10⁶ cells per condition improves analytical robustness compared with 1 × 10⁶ cells, which showed greater dispersion in repeated experiments. Additional critical refinements included the use of cold methanol to enhance protein precipitation efficiency and the incorporation of an internal standard to monitor and minimize procedural variation. The method enables sensitive and precise quantification of intracellular VCR across relevant concentration ranges, although the lowest tested dose (0.01 µM) was not quantifiable after the 3 h incubation. Overall, this micromethod provides a practical, reproducible, and scalable approach for measuring intracellular VCR in primary ALL samples. Its design supports comparative analyses, validation of ex vivo models, and future methodological applications aimed at integrating intracellular drug quantification into broader pharmacological assessments.

Introduction

Acute lymphoblastic leukemia (ALL) accounts for approximately 25% of all childhood cancers and remains the most common pediatric malignancy^1,2. Although advances in therapy have markedly improved survival rates, now exceeding 90%-the biological complexity of ALL continues to drive efforts to develop tools that better characterize the disease at the cellular level³.

Vinca alkaloids, particularly VCR, are key components of ALL treatment protocols⁴. Because VCR exerts its cytotoxic effects intracellularly, understanding its uptake and accumulation in leukemic cells is fundamental for studies involving drug response, pharmacodynamics, and cellular behavior in ex vivo systems. However, most available approaches for assessing intracellular drug levels rely on indirect measurements or semi-quantitative methods, limiting reproducibility and sensitivity^5,6,7.

In parallel, many investigations still depend heavily on leukemia cell lines, which lack the biological heterogeneity and clinical relevance of patient-derived primary cells. Primary samples often present challenges such as limited cell numbers, variable sample quality, and the need for highly sensitive analytical techniques capable of detecting low-abundance intracellular compounds⁸.

Liquid chromatography-tandem mass spectrometry (LC-MS/MS) provides the specificity and sensitivity required for precise quantification of chemotherapeutic agents, yet standardized protocols for measuring intracellular VCR directly in primary leukemic cells remain scarce. Establishing robust micromethods compatible with limited patient material may facilitate a broad range of applications, including pharmacological profiling, evaluation of drug-cell interactions, and comparative studies across disease subtypes or therapeutic conditions.

Here in this protocol, we describe the development and optimization of a micromethod for ex vivo quantification of intracellular VCR in primary leukemic cells using LC-MS/MS. To obtain sufficient material for method development, patient samples were transplanted into immunodeficient NSG mice to generate PDX models⁹. After in vivo expansion, human leukemic cells were isolated and cryopreserved¹⁰. After, they were thawed, incubated with VCR, washed, lysed, and analyzed by LC-MS/MS to determine intracellular drug levels.

This method offers several advantages: it requires a small number of cells (2.5 million), is sensitive and rapid, and is compatible with clinically relevant primary samples. Because it directly measures intracellular drug content, it provides a reliable quantitative tool for researchers investigating drug uptake, cellular responses to chemotherapeutics, or other pharmacological questions in hematologic malignancies.

Overall, this protocol expands the analytical toolkit available for studying pediatric ALL and can be readily adapted for other drugs or sample types, supporting future investigations into leukemia biology, drug behavior, and precision pharmacology.

Protocol

The leukemia samples used in this study were obtained from bio-banked vials of patients treated at Centro Infantil Boldrini, who allowed the use of their cells through an informed consent form. The projet was approved by the Institutional Ethics Committee (CAAE 34601120.7.0000.5376). The protocols for the generation of PDX in animals were carried out in accordance with the regulations and ethical guidelines of the Animal Ethics Committee (Comissão de Ética no Uso de Animais) of Centro Infantil Boldrini (CEUA/Boldrini 0011-2020).

1. Sample preparation and drug incubation (Day 1)

1. Preparation of stock and working solutions
   1. Weigh 2.5 mg of Vincristine sulfate (molecular weight: 923 g/mol) and dissolve it in 2.0 mL of dimethyl sulfoxide (DMSO) in a 2.0 mL microtube to prepare a 1.354 mM stock solution. Vortex until fully dissolved.
      ​CAUTION: DMSO is an irritant; wear gloves and handle it in a chemical fume hood.
   2. Prepare the 100 µM working solution by mixing 147.71 µL of the VCR stock solution with 1,852.28 µL of DMSO in a 2.0 mL microtube. Vortex thoroughly.
   3. Distribute 500 µL of the working solution into four 0.6 mL microtubes and store at -20 °C for up to several months. Avoid thawing the aliquots more than three times.
   4. Prepare a 100 µM vinblastine (VINB) working solution by mixing 181.81 µL of a 1.1 mM stock solution (Vinblastine sulfate 1 mg/mL, molecular weight: 909.07 g/mol) with 1,818.19 µL of sterile ultrapure water in a 2.0 mL microtube. Vortex until homogeneous.
   5. Aliquot the VINB working solution the same way as done for VCR.
   6. Prepare methanol + 0.1 µM VINB solution by mixing 49.95 mL of methanol with 50 µL of the VINB working solution in a 50 mL tube. Vortex until homogeneous. Store it at -20 °C up to 3 months.
      CAUTION: Methanol is toxic and flammable; handle with appropriate PPE in a fume hood.
   7. Prepare a water + 0.1% formic acid solution by mixing 49.95 mL of ultrapure water with 50 µL of formic acid in a 50 mL tube. Vortex well. Store at room temperature (RT).
      CAUTION: Formic acid is corrosive; handle with gloves and eye protection.
2. Thawing and washing primary cells
   1. Thaw cryopreserved cell samples by gently swirling the vial in a beaker containing 15 mL of 37 °C distilled water until fully thawed.
      ​NOTE: Information regarding the cell collection and freezing protocols can be found in previous publications^9,10.
   2. Transfer the contents of the vial to a 50 mL conical tube containing 10-15 mL of sterile 1x PBS with a sterile disposable Pasteur pipette. Centrifuge at 300 × g for 5 min at RT.
   3. Discard the supernatant by inverting the tube.
   4. Resuspend the pellet in 10 mL of AIM V medium with a disposable Pasteur pipette. Count viable cells and measure average cell size using an automated counter.
      NOTE: Using cells that exhibit at least 80% viability relative to the total number of cells collected is recommended.
   5. Calculate the volume needed to collect 2.5 × 10⁶ viable cells using a simple proportion based on the measured concentration. For example, if the concentration is 5 × 10⁶ cells/mL, the required volume is 0.5 mL.
      ​NOTE: Perform each treatment in triplicate for data reproducibility. Save the medium size of the cells provided in the automated cell counter; use it to calculate the volume of the cells.
3. Drug treatment setup
   1. Calculate the volume of VCR working solution required to reach a final concentration of 1 µM in a total volume of 2.0 mL. Vortex the stock solution before pipetting.
   2. Add AIM V medium, then 2.5 × 10⁶ cells, and finally the VCR solution to a 2.0 mL microtube. Vortex briefly and spin down lightly (5 s) to collect any liquid from the tube cap.
   3. Incubate the open tubes at 37 °C, 5% CO₂ for 3 h.
   4. After incubation, close the lids and centrifuge the samples at 300 × g for 5 min at RT.
   5. Carefully discard the supernatant using a pipette, then add 1 mL of PBS at RT to the tube.
   6. Repeat the centrifugation at 300 × g for 5 min at RT, supernatant removal, and PBS wash two additional times, for a total of three washes.
   7. Carefully remove all supernatant using a pipette, resuspend the pellet in 100 µL of PBS, and vortex until the pellet is fully resuspended.
   8. Add 400 µL of cold methanol + 0.1 µM vinblastine (-20 °C) directly to the sample, avoiding contact with the tube walls.
   9. Store the samples overnight at -80 °C.
      ​NOTE: Samples can be stored at -80 °C for a few weeks (safe stop).

2. Sample processing for LC-MS/MS (Day 2)

1. Sample extraction
   1. Pre-cool the centrifuge to 4 °C before removing samples from the -80 °C freezer.
   2. Immediately after taking the samples from the freezer, centrifuge them at 13,000 × g for 20 min at 4 °C.
   3. Transfer 300 µL of supernatant to a 1.5 mL microtube.
   4. Dry the samples completely using a vacuum concentrator at 40-45 °C for 1 h and 40 min.
      NOTE: Dried pellets can be stored at -20 °C for a few weeks (safe stop).
   5. Resuspend each pellet in 400 µL of water + 0.1% formic acid (previously prepared). Vortex gently until the pellet is fully dissolved.
   6. Centrifuge the samples again at 13,000 × g for 20 min at 4 °C.
   7. Transfer 300 µL of supernatant to a 2.0 mL screw-top vial for LC-MS/MS analysis.

3. LC-MS/MS setup and data acquisition

1. Mobile phase preparation and column conditioning
   1. Prepare Solvent A by mixing ultrapure water with 0.1% formic acid.
   2. Prepare Solvent B by mixing acetonitrile with 0.1% formic acid.
      CAUTION: Acetonitrile is toxic and flammable; handle in a chemical fume hood.
   3. Sonicate each solvent in an ultrasonic bath for 5 min. Connect the bottles to the pump module and purge the system at high flow to remove contaminants.
   4. Install a reversed-phase BEH C18 column (1.7 µm, 2.1 mm × 50 mm).
      NOTE: To perform the analysis, this study used an Acquity H-Class chromatographic system coupled to a Xevo TQS-Micro triple-quadrupole mass spectrometer.
   5. Use a binary gradient composed of Solvents A and B, both previously prepared.
   6. Run the chromatographic gradient from 5% to 95% of Solvent B over the first 2 min, followed by 2 min of Solvent A for column washing and re-equilibration.
   7. Set the flow rate to 0.2 mL/min throughout the 4-min run. Maintain the column temperature at 40 °C and inject 10 µL of each sample.
2. LC-MS/MS method setup
   1. Open the LC-MS/MS software. Click File > New > MS Method to start a new method.
   2. Go to the Function tab and set the function type to multiple reaction monitoring (MRM). Enter ion transitions and parameters listed in Table 1 for VCR and VINB (internal standard).
   3. Set the Acquisition Time to 4 min. Click Apply to save.
      NOTE: Do not exceed 4 min to ensure column re-equilibration between injections.
   4. Configure UHPLC parameters under Inlet Method > Edit LC Method and save as a .mth file (Table 2).
   5. Set source parameters under the Tune tab according to Table 3. Save as a .tun file.
   6. Save the complete method: File > Save As, and Export as a .mth file.
3. Creating a sample list and starting the run
   1. In the software's home screen, open Sample List and create a list with the sample names, injection volumes, vial positions, and the method to be used.
   2. Place a vial with ultrapure water in the autosampler tray and designate it as a blank. Run the blank between every set of triplicate injections to avoid carryover.
      NOTE: Before starting the run, ensure the column pressure (ΔP) is stable and less than 10 psi.
   3. Select the desired rows in the Sample List and click Run > OK to begin data acquisition.

4. Data analysis

1. Data visualization and export
   1. After the run completes, view chromatograms by selecting a sample and clicking Chromatogram in the top menu bar.
   2. On the home screen, click on TargetLynx, select the files that just ran, and click Process > Select the vincristine method > OK, to generate output tables.
   3. Click Report > Export Table and save the processed data as a .csv or .xlsx file.
      NOTE: Export raw peak areas for both the analyte and internal standard.
2. Data organization and ratio calculation
   1. Open the exported file and create the following columns:
      1. Sample: Add the sample or vial name.
      2. Run: Indicate which replicate the value corresponds to.
      3. VCR Area: Input the area under the peak for vincristine.
      4. VINB Area: Input the area under the peak for vinblastine.
      5. VCR/VINB Ratio: Normalize each VCR value by dividing it by its corresponding VINB value.
   2. Add columns for standard deviation values of VCR area, VINB area, and VCR/VINB ratio for each triplicate set.
      ​NOTE: Normalization using the internal standard allows correction for variation in extraction or injection volume.
3. Plotting results
   1. Open the preferred graphing software and select the Grouped Table format.
   2. Choose Enter or import data into a new table, and under Options, select Enter 3 replicate values in side-by-side subcolumns.
   3. Plot only the normalized VCR/VINB ratios for each sample, omitting raw area values.

Results

Representative chromatographic results obtained after intracellular accumulation assays using primary ALL PDX cells incubated with VCR and VINB are shown in Figure 1. Samples were prepared using 2.5 × 10⁶ cells per condition. Under the chromatographic conditions described, VCR was consistently retained at 2.85 min with minimal matrix effect, and peak shape was symmetrical and well-resolved. These profiles illustrate the expected chromatographic behavior of VCR and VINB when the pr...

Discussion

Accurate assessment of intracellular drug levels in primary leukemic cells is essential for understanding drug handling at the cellular level and for supporting the development of functional assays that complement molecular profiling. Existing analytical approaches often require large numbers of cells, involve lengthy processing steps, or rely on indirect readouts that do not reflect the true intracellular availability of chemotherapeutic agents^5,6. These limitat...

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
2.0 mL screw-top vial	Flowsupply	8-2400	Used with glass inserts with spring (5 x 29 mm) from the same company (Cat. 5-29015)
2.0 mL screw-top vial	Flowsupply	8-2400	Used with glass inserts with spring (5 x 29 mm) from the same company (Cat. 5-29015)
Acetonitrile	Merck (Sigma-Aldrich)	100030
Acetonitrile	Merck (Sigma-Aldrich)	24010152
AIM V	Thermo Fisher Scientific	12055091
Chromatographic system Acquity UPLC H-Class	Waters Co.	https://www.waters.com/nextgen/br/pt/products/chromatography/chromatography-systems/acquity-uplc-h-class-plus-system.html?srsltid=AfmBOoo0mjNgaucBI-Exv97cEmWE96_lVkqKi2IR0IPtA-h1feG-WvtW
Countess 3 FL	Thermo Fisher Scientific	A49892
DMSO	Merck (Sigma-Aldrich)	D 8418
Falcon 50 mL	Sarstedt	62.547.254
Formic acid	Merck (Sigma-Aldrich)	F0507
Glass bottle	Merck (Sigma-Aldrich)	41121800
Incubator	PHCBI	MCO-50M-PE
Methanol	Merck (Sigma-Aldrich)	34860
Microtube 1.5 mL	Eppendorf	41121702
Microtube 2.0 mL	Eppendorf	30120094
PBS	Merck (Sigma-Aldrich)	PPB006
Speevac miVac	Fisher Scientific	56340
The software for data acquisition	Waters Co.	MassLynx
Triple quadrupole mass spectrometer Xevo TQ-S micro	Waters Co.	https://www.waters.com/nextgen/us/en/products/mass-spectrometry/mass-spectrometry-systems/xevo-tq-s-micro.html?srsltid=AfmBOoqqmLRNfHGqvtnwaL7xYOOEXPV3NJ9qYNH9HAwqXKXrMvLRQXSW
Ultrasonic Sonicator	Quimis	Q335D
Vinblastine 1 mg/mL	Libbs	https://www.libbs.com.br/nossos-produtos/antineoplasico-faulblastina/	Vinblastine sulfate
Vincristine	Cayman Chemicals	11764