72
A. Dal Pozzo et al. / Bioorg. Med. Chem. 18 (2010) 64–72
in new eppendorf tubes and processed adding 200
l
L of methanol/
were kept on ice for 10 min and then centrifuged at 4000g for
10 min at 4 °C. Supernatants were analyzed by HPLC-FL as de-
scribed above. All data were expressed as mean (ng of drug/mL
plasma or ng/g tissue) SE.
acetonitrile mixture (1:1 v/v). After vortexing, samples were placed
on ice for 10 min, then centrifuged at 14,000g for 200 at 4 °C and
supernatants were transferred into auto-sampler vials. Protein
concentration of cell lysates was determined using Comassie
(Bradford) Protein Assay kit (Pierce). Drug containing cell culture
Acknowledgements
medium was processed as described adding 700 lL of extraction
mixture. Supernatants were analyzed by HPLC-FL (Beckman Instru-
ments) and analytical data were acquired and processed by a com-
puter system (32Karat Software, Beckman-Coulter). The separation
was performed by isocratic elution (30% CH3CN, 70% 0.1 M AcOH
containing 0.1%TEA, pH ꢃ3.5) with a flow rate of 1 mL/min on a
This work was supported by Sigma-Tau, Roma, Italy. We thank
Patrizia Tobia and Matilde Cati for technical assistance.
Supplementary data
discovery HS-F5 column, 100 ꢀ 4.6 mm,
5 lm (Supelco). The
Figures S1 and S2 describing decreasing rates of 8a at different
pHs. Supplementary data associated with this article can be found,
detection was performed with a spectrofluorometric detector
(RF-10AXL, Shimadzu) at kex 370 nm and kem 510 nm. All data were
expressed as mean (nmol of drug/mg of total proteins) SE.
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