SUPPLEMENTARY INFORMATION
Bioanalytical method
Instrumentation and chromatographic conditions
The liquid chromatographic system consisted of a Prominence Ultra-Fast Liquid Chromatograph
(UFLCXR) equipped with a binary pump (LC-20ADXR), an autosampler (SIL20ACXR), a column oven
(CTO-20 AC), and a solvent degasser (DGU-20A3), all made by Shimadzu U.S.A. Manufacturing, Inc
(Canby, OR, USA). The mobile phase consisted of (A) 0.1% formic acid in water and (B) 0.1% formic acid
in acetonitrile, pumped at a flow rate of 0.25 mL/min. The chromatographic separation was performed on
a Phenomenex Luna C18 column (3 µm, 100 A˚ 50 × 2.00 mm) using a gradient elution, which was as
follows: 0.0→3.5 min, B% 0→80; 3.5→5.5 min, B% 80→80; 5.5→7.5 min, B% 80→0.0; 7.5–9.5 min, B%
0.0→0.0. The column and autosampler temperatures were maintained at 25 ˚C. The injection volume was
3 μL and the total runtime for each injection was 9.5 min.
An API-4000 LC-MS/MS mass spectrometer system (AB SCIEX, Framingham, MA, USA) equipped with
Turbo VTM ion source was used for mass analysis and detection. Mass spectrometric analysis was
performed in the positive ionization mode with a dwell time of 250 ms. Multiple reaction monitoring (MRM)
of the precursor-product ion transitions m/z 304.10 > 170.00 for clofarabine and m/z 286.00 > 170.00 for
cladribine (internal standard, IS) was used for quantification. The ion spray voltage and temperature were
set at 5000 V and 500 ˚C, respectively. The source/gas parameters, viz. the collision gas (CAD), curtain
gas (CUR), ion source gas 1 (GS1) and ion source gas 2 (GS2) were set at 5, 10, 20 and 35 psi. The
compound parameters, viz. the declustering potential (DP), entrance potential (EP), and collision cell exit
potential (CXP), were optimized to 60, 10 and 15 V for both clofarabine and cladribine. The collision
energy (CE) was set at 29 and 23 for CF and IS, respectively. MS data acquisition was performed with
Analyst 1.5.2 ® software and processed (integrated) with MultiQuant 2.1.1 software (AB SCIEX,
Framingham, MA, USA)
Quantification of clofarabine
Calibration standards were prepared at 1, 10, 50, 100, 200, 301, 406 and 508 ng/mL in mouse plasma
and aCSF. Protein precipitation with acetonitrile and liquid-liquid extraction with ethyl acetate was used
for the plasma and aCSF sample preparation, respectively. Protein precipitation: an aliquot of 25 µL of
mouse plasma was mixed with 10 µL of IS working solution (100 ng/mL) in a 1.5 mL eppendorf tube. To
this, 80 µL of 0.1% formic acid containing acetonitrile was added and the resulting mixture was vortexed
for 3 min using multi-tube votexer (Fisher Scientific, Pittsburgh, PA, USA). Following centrifugation for 5
min at 10000 rpm using Sorvall ST40R centrifuge (Thermo Scientific, Asheville, NC, USA), an aliquot of
the clear supernatant was transferred into autosampler vial, and 3 µL aliquot was injected into the
chromatographic system. Liquid-liquid extraction: an aliquot of 10 µL of IS working solution (100 ng/mL)
was added to 25 µL of aCSF in a 1.5 mL eppendorf tube. To this, 700 µL of ethyl acetate was added and
vortexed for 10 min. The samples were centrifuged at 10000 rpm for 5 min. The organic layer was then
transferred to a glass vial and dried in a CentriVap Console (Labconco, Kansas City, MO, USA) at 40 °C
for 20 min. The dried extracts were then reconstituted with 80 µL of 0.1% formic acid in water and 0.1%
formic acid in acetonitrile (20:80, v/v) and an aliquot of 3 µL was injected onto the chromatographic
system. The retention times of clofarabine and IS were found to be 3.1 ± 0.1 min and 3.0 ± 0.1 min,
respectively. There were no endogenous interferences at the retention times of clofarabine and IS. The
developed method was found to be linear and reproducible with typical r > 0.99, in both matrices (plasma
and aCSF). Precision and accuracies were assessed by running six replicates of high, medium, low and
lower limit of quantification quality control samples in the relevant matrix at 440 ng/mL, 150 ng/mL, 3
ng/mL and 1 ng/mL, respectively. The intra- and inter- day assay coefficients of variations were found to
be < 5.08% and accuracies were found to be between 86% - 109%.
Figure Legends
Supplementary Figure 1: Clofarabine in vitro washout study In vitro washout study of
clofarabine on mouse ependymoma cells. Cells were incubated for indicated time period with
clofarabine and then to a total of 72 hrs. (Error bars represent standard deviation)
Supplementary Figure 1