Sample preparation method for determination of Axitinib in plasma using ISOLUTE® SLE+

Introduction

Axitinib (Figure 1) is a type of molecularly targeted drug used in renal cell carcinoma. To avoid serious side effects and achieve maximal therapeutic efficacy, it is crucial to determine the correlation between side effects on blood levels and treatment efficacy. Therapeutic Drug Monitoring (TDM) is one of the measures to achieve this.

In this application note, ISOLUTE® SLE+ was used as a sample preparation cartridge for Axitinib determination in plasma using the principles of supported liquid extraction (SLE).

ISOLUTE® SLE+ Supported Liquid Extraction plates and cartridges offer an efficient alternative to traditional liquid-liquid extraction (LLE) for bioanalytical sample preparation, providing high analyte recoveries, no emulsion formation, and significantly reduced sample preparation time.

Figure 1. Structural formula of Axitinib

Analytes

Axitinib (Axitinib, CAS:319460-85-0)

Internal standards

Osimertinib (Osimertinib, CAS: 1421373-65-0)

Sample preparation procedure

Format

ISOLUTE® SLE+ 400 µL Sample Volume cartridges, (part number: 820-0055-B).
An equivalent 96-well plate format, (part number 820-0400-P01) is also available.

Sample pre-treatment

To plasma (200 μL), add 5.0 ng/mL of Osimertinib as an internal standard. Add water (HPLC grade or higher) (200 μL) and vortex mix for 30 seconds.

Sample loading

Load 400 μL of the sample solution onto the ISOLUTE® SLE+ cartridge and apply light pressure (3 psi) or vacuum (-0.2 bar) to initiate flow. Allow the sample to absorb for at least 5 minutes and wait for samples to stabilize. Ensure that all sample solution is absorbed onto the diatomaceous earth.

Sample elution

Add 900 μL of methyl tert-butyl ether (MTBE) and allow to flow under gravity for 5 minutes. Then, add another 900 μL of the solvent, and allow to stand for 5 minutes or more. If required, the extraction can be completed with application of gentle pressure (3 psi) or vacuum (-0.2 bar) (10 to 30 seconds).

Evaporation and reconstitution

Evaporate the extract with a nitrogen gas evaporator, and reconstitute the extract with water: methanol (1:1, v/v, 2 mL).

UHPLC conditions

Instrument

Nexera LC-30AD (Shimadzu)

Column

ACQUITY UPLC® BEH C18 1.7 μm (2.1 mm × 50 mm column; Waters)

Mobile phase

A: 10 mmol/L ammonium acetate aqueous solution
B: acetonitrile 

Flow rate

0.4 mL/min 

Gradient conditions 

Column temperature

40 °C

Injection volume

1 µL

Mass spectrometry conditions

Instrument

LCMS-8060 (Shimadzu)

Ionization mode

ESI positive

Nebulizer gas flow rate

3.00 L/min

Drying gas flow rate

10.00 L/min

Heating gas flow rate

10.00 L/min

Interface temperature

350 ⁰C

DL temperature

200 ⁰C

Heat block temperature

350 ⁰C

CID gas

270 kPa

SRM transitions

Axitinib: m/z 387.40 > 356.05, Rt 2. 14 min, Collision Energy -20
Osimertinib (IS): m/z 500.30 > 72.15, Rt 2. 43 min, Collision Energy -27

Results

SRM (Selected Reaction Monitoring) chromatograms of Axitinib and Osimertinib (IS) are shown in Figure 2. Under the LC conditions used in this application note, two peaks were detected in the Axitinib chromatogram. This was attributed to the separation of the isomers. In this study, we determined the peak area value for the peak at elution time of 2.14 minutes and used this to create the calibration curve*. The calibration curve generated is shown in Figure 3.

Figure 2. SRM chromatograms of Axitinib (top) and Osimertinib (IS) (bottom)

Figure 3. Standard curve for Axitinib

Axitinib has a wide dynamic range of 0.01 to 25 ng/mL covering the range of blood concentrations required for TDM (0.1 to 10 ng/mL) and has a good linearity with a multiple correlation coefficient (r2) of 0.9999 or more.

(* Reference: Biomedical Chromatography. 2018;32:e4147)

Confirmation of analyte recovery and matrix factors

Sample pre-treatment is very important in samples of biological origin. ISOLUTE® SLE+ supported liquid extraction cartridges were used to eliminate the effects of matrix components such as proteins, phospholipids, and salts and to allow improved quantitative analyses. Figure 4 shows the SRM chromatograms obtained by SLE pre-treatment of the control plasma spiked with 50.0 ng/mL axitinib. No interference from contaminants in the plasma was observed. Recovery rates and matrix factors after SLE pre-treatment for 3 axitinib plasma concentrations (0.5, 5.0, and 20.0 μg/mL) are shown in Table 1. Recovery was calculated by comparing the peak area of samples spiked with axitinib pre-extraction (A) with that of blank samples spiked after extraction (B).

Matrix factors were calculated by comparing the area values of (B) and the standard solution (S). As a result, a recovery rate of more than 95% was obtained at each concentration, and the values of matrix factors were sufficiently low. It was quantitatively confirmed that ISOLUTE SLE+ pre-treatment effectively eliminates matrix effects. In this application note, we recommend the use of Osimertinib as IS for axitinib, but use of stable isotope dilution as an internal standardization approach would also be appropriate.

* Recovery rate = [A]/[B] × 100; Matrix Factor = 1-[B]/[S] × 100

Figure 4. SRM chromatograms of 50.0 ng/mL Axitinib in plasma after ISOLUTE® SLE+ extraction.

Ordering information

This application note was prepared in collaboration with the Pharmaceutical Department of Gunma University Hospital, Japan.

Literature number: AN937