Mitoxantrone Quantification by HPLC-MS/MS in Caco-2 Culture Media

Abstract

Mitoxantrone is a marker substrate of breast cancer resistance protein (BCRP). BCRP is involved in a number of pharmacokinetic drug-drug interactions. The transporter’s possible saturability makes it advisable to use low concentrations of mitoxantrone for in vitro studies. Consequently, mitoxantrone quantification requires a method with high sensitivity.The aim of the study was to develop and validate a procedure for mitoxantrone quantification in Caco-2 culture media by HPLC-MS/MS.Materials and methods. The authors used an Ultimate 3000 HPLC system and a TSQ Fortis triple quadrupole mass spectrometer by Thermo Fisher Scientific and a Selectra C18 column (4.6×100 mm, 5 μm, 100 Å) by United Chemical Technologies. The elution ran in a gradient mode with a mobile phase of 1% formic acid solution and methanol. Experimental parameters were as follows: eluent flow rate, 0.3 mL/min; separation column temperature, 35 °C; injection volume, 5 μL; ana lysis time, 10 min; approximate mitoxantrone retention time, 5.51 min. The sample preparation involved protein precipitation from the culture medium with methanol, followed by centrifugation at 13,000 g for 10 min. The detection was performed using electrospray ionisation in the positive ion mode. Detection parameters were as follows: electrospray voltage, 3700 V; sheath gas flow rate, 50 L/min; auxiliary gas flow rate, 10 L/min; sweep gas flow rate, 1 L/min; ion-transfer tube temperature, 300 °C; and evaporator temperature, 350 °C. The detection was set at mass transitions of m/z 455 to 88.2 and m/z 455 to 358.1, with the collision energy for these transitions amounting to 25 V and 18 V, respectively. The source fragmentation was at 0, and the CID gas pressure was at 2 mTorr.Results. The analytical procedure showed selectivity, high sensitivity (limit of detection, 10 nmol/L; lower limit of quantification, 50 nmol/L), accuracy, precision, and linearity in the concentration range of 50–1000 nmol/L. The authors observed no carryover or matrix effects. A simulation of real-life storage conditions demonstrated high stability of mitoxantrone samples. Thus, the analytical procedure enables preclinical evaluation of medicinal product effects on the functional activity of BCRP, based on assessing the transcellular mitoxantrone transport in the presence of a test product.Conclusion. The authors developed and validated the analytical procedure for mitoxantrone quantification in Caco-2 culture media by HPLC-MS/MS.

Keywords

• mitoxantrone
• hplc-ms/ms
• bcrp marker substrate
• validation
• preclinical studies