Supplemental Digital Content 1
Chemicals and Reagents
Lenalidomide and the internal standard umbelliferone were purchased from Toronto
Research Chemicals Inc. (Ontario, Canada) and Tokyo Chemical Industry Co., Ltd.
(Tokyo, Japan), respectively. Oasis hydrophilic lipophilic balance (HLB) extraction
cartridges (1 mL, 30 mg) were purchased from Waters (Milford, MA, USA). All other
reagents were purchased from Nacalai Tesque (Kyoto, Japan). All solvents were
liquid-chromatography grade. Stock solutions of lenalidomide and umbelliferone were
prepared by dissolving the dry reagents in dimethylsulfoxide and methanol, respectively,
to yield concentrations of 1.0 mg/mL.
Analytical methods
A 10-μL solution of umbelliferone (10 ng), as an internal standard for quantitation of
lenalidomide, was added to the 100 μL plasma sample, followed by dilution of the
sample with 900 μL of water and vortexing for 30 s. This mixture was applied to an
Oasis HLB extraction cartridge that had been previously activated with methanol and
water (1.0 mL each). The cartridge was then washed with 1.0 mL of water and 1.0 mL
of 10% methanol in water followed by elution with 1.0 mL of 100% methanol. The
eluate was evaporated to dryness under vacuum at 65°C by using a rotary evaporator
(Iwaki, Tokyo, Japan). The mean extraction recovery value of lenalidomide was 96.8%
in the concentration range of 1.0 to 2,000 ng/mL. The residue was dissolved in 20 μL of
methanol (vortex-mixed for 30 s), and a 1-μL aliquot of the sample was then processed
on the liquid chromatography-tandem mass spectrometry (LC–MS/MS) system
equipped with electrospray ionization (ESI) interface operated in the positive mode
(LCMS-8030, Shimadzu Corporation, Kyoto, Japan). The chromatographic separation
was achieved by LC using a Shim-pack XR-ODS II column (75 mm L × 2.0 mm I.D.;
Shimadzu Corporation, Kyoto, Japan) operated at 40°C. The gradient mobile phase
consisted of 0.1% acetic acid in water and methanol at a flow rate of 0.2 mL/min: 5%
methanol (run time: 0 min) → 80% (5 min) → 100% (5.01–7 min) → 5% (7.01–10 min).
The precursor to product ion transitions of m/z 260.10 > 149.00 and m/z 163.05 >
106.95 were used to quantify lenalidomide and umbelliferone, respectively. Calibration
curves for lenalidomide in plasma were linear over a concentration range of 1.0 to 2,000
ng/mL, with the limit of quantitation being 1.0 ng/mL. The coefficients of variation for
intra- and inter-day assays were less than 11.6%. Accuracies for intra- and inter-day
assays were within 4.8%.