Experimental section

3.2.1. Chemicals

The etoposide standard and teniposide (internal standard) were purchased from the Sigma-Aldrich Co. (St. Louis, MO, USA); 3’-O-demethyl etoposide (etoposide catechol)

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was synthesized by Toronto Research Chemicals Inc. (North York, Canada). MS-grade methanol was purchased from Scharlau Chemie (Sentmenat, Barcelona, Spain).

Acetonitrile (ACN) was obtained from J.T. Baker (Phillipsburg, NJ), and formic acid solution (99%) was purchased from Sigma (St. Louis, MO, USA).

3.2.2. Preparation of the standard, CSF, and plasma samples

Etoposide, etoposide catechol, and teniposide (PCI-IS) stock solutions were prepared separately in methanol at concentrations of 100 g mL⁻¹. The working solutions were prepared by spiking appropriate amounts of etoposide, etoposide catechol, and teniposide stock solutions into deionized (DI) water to obtain diluted working solutions of 1000 ng mL⁻¹.

A 40-L aliquot of plasma was diluted with 160 μL of methanol. The deproteinized

sample was centrifuged at 10,000 x g for 15 min, and the supernatant was then filtered through a 0.22-μm syringe filter (RC-4, Sartorius, Göttingen, Germany) prior to the LC-ESI-MS analysis. For CSF sample preparation, 200 μL of CSF was directly filtered through a 0.22-μm syringe filter (RC-4, Sartorius, Göttingen, Germany) prior to the LC-ESI-MS analysis.

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3.2.3. UPLC-ESI-MS system

The instrument used was an Agilent 1290 U-HPLC system coupled with an Agilent 6460 triple quadrupole mass spectrometer (Agilent Technologies, Santa Clara, CA). The

injection volume was 5 μL. The separation was performed using a Kinetex™ column (2.1 × 50 mm, 2.6 μm) (Phenomenex, Torrance, CA). The analytical column was

maintained at 40 °C. The mobile phase consisted of 0.1% aqueous formic acid (solvent A) and 0.1% formic acid in ACN (solvent B). A 0.3-mL min^-1 linear elution gradient was used: 0-2 min, 20-95% B; 2-3 min, 95% B; and 3-4.5 min, column re-equilibration with 20% B. The sample reservoir was maintained at 4 °C. The JetStream electrospray ionizer was employed as the ion source. The MS parameters were set as follows: 325 °C for the drying gas temperature, 6 L min⁻¹ for the drying gas flow, 40 psi for the nebulizer flow, 375 °C for the sheath gas temperature, 8 L min^-1 for the sheath gas flow, and 4000 and 3500 V for the capillary voltage in the positive and negative modes,

respectively. The mass spectrometer was configured in the multiple reaction monitoring mode and monitored the transitions of m/z 575.2 → 229 and 575.2 → 185.1 for etoposide catechol, 589.2 → 229 and 589.2 → 185 for etoposide, and 657.2 → 228.9

for teniposide (PCI-IS). Teniposide was selected as the PCI-IS and was dissolved in

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ACN at a concentration of 100 ng mL⁻¹ and then introduced into the LC eluent at a flow rate of 0.1 mL min^-1.

3.2.4. The use of the PCI-IS method in combination with matrix normalization factors

(MNFs) for the correction of matrix effects

All of the data obtained using the Agilent triple quadruple spectrometer were converted into the format of comma-separated values (csv) and were processed with Microsoft Excel 2007 (Albuquerque, NM, USA). The information in the csv file included the mass transition, retention time, and signal intensity. The MS acquisition rate was set to 1 spectrum s^-1. The PCI-IS method assumed that the response ratio of target analyte to PCI-IS was proportional to the concentration of the target analyte. The analyte to PCI-IS response ratios obtained in the standard, plasma, and CSF at each time point were used to calculate the MNFs.

One plasma and one CSF sample spiked with the same concentrations of etoposide and etoposide catechol were used to calculate the MNFSTD-plasma and the MNFSTD-CSF

values. Because the individual differences between the samples would be corrected for by the PCI-IS, there are no requirements for the selection of the plasma and CSF

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samples used to calculate the MNFs. The ratio of the etoposide and etoposide catechol responses to the teniposide (PCI-IS) response in the plasma or CSF samples at each time point was divided by the ratio of the etoposide and etoposide catechol responses to the teniposide responses in the standard solutions at each time point to obtain the MNFSTD-plasma and MNFSTD-CSF ratios. Lastly, the ratio of the etoposide and etoposide catechol response to the teniposide response in every plasma (or CSF) sample at each time point was divided by MNFSTD-plasma (or MNFSTD-CSF) at the identical retention time, and the corrected ratios were used to generate the new corrected chromatograms for the quantification of etoposide and etoposide catechol.

3.2.5. Validation

3.2.5.1. Linearity, limits of detection (LODs), and limits of quantification (LOQs)

Aliquots of the etoposide and etoposide catechol stock solutions were added to deionized water to obtain 10, 50, 150, 250, and 500 ng mL⁻¹ etoposide and etoposide catechol standard solutions to construct the calibration curves used for the quantification of etoposide and etoposide catechol in plasma and CSF. The calibration curves were established by plotting the ratios of etoposide and etoposide catechol with teniposide (PCI-IS) against the etoposide or etoposide catechol concentrations.

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The limit of detection (LOD) of each analyte was determined as the concentration at which the signal to noise ratio equals 3 (S/N = 3). The limit of quantification (LOQ) of each analyte was determined as the concentration at which the signal to noise ratio equals 10 (S/N = 10).

3.2.5.2. Accuracy and precision

For testing the precision and accuracy of the method, aliquots of etoposide and etoposide catechol were spiked into 7 blank plasma and 3 blank CSF samples to obtain 10, 150, 500 ng mL⁻¹ etoposide and etoposide catechol spiked samples, and they were tested 4 times a day for 3 days. The obtained data were corrected by the PCI-IS and MNFs and were quantified by the standard curve established using the standard

solutions.

3.2.6. Protein analysis

The CSF and plasma samples were deproteinized using different volumes of methanol or acetonitrile and then centrifuged (10,000 x g for 15 min) to precipitate the denatured protein. The residual protein in the supernatant was reconstituted using a

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0.1% Triton X-100 solution, and 2 μL of 6-fold sample buffer (containing 60 mM Tris-Cl, 2% SDS, 10% glycerol, 5% β-mercaptoethanol, and 0.01% bromophenol blue pH = 6.8) was added to the lysed sample. The mixtures were heated in a water bath at 95 °C for 5 min to denature the protein into negatively charged linear structures and then quickly put on ice for 10 min to avoid protein annealing. A 10-μL aliquot of the protein extracts was separated by 10% SDS-PAGE at 100 V for 180 min. The gel was carefully rinsed with deionized water and fixed with 7% acetic acid in 40% MeOH for 1 hr. The gels were further stained with Coomassie blue reagent (Sigma, St. Louis, MO) for 2 hr and then destained with 10% acetic acid in 25% MeOH for 60 sec. We further rinsed the gel with 25% MeOH until the background was clear for observation.

3.2.7. Collection of clinical samples

All of the plasma and CSF samples were obtained from National Taiwan University Hospital. The local ethics committee approved this study, and signed inform consent was received from the patients that participated. Etoposide was intravenous administered at a dose of 70 mg m^-2 to patients. Avastin® (bevacizumab) was intravenous administered at a dose of 15 mg kg^-1 before the second etoposide treatment.

The blood samples were collected in EDTA-containing tubes. The blood samples were

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centrifuged at 10,000 x g for 15 min, and the plasma samples were stored at -80 ^◦C until use. The CSF samples were collected in centrifuge tubes. The collected CSF samples were stored at -80 ^◦C until use.