A rapid, simple and reliable HPLC-triple quadrupole tandem mass spectrometer method for a simultaneous quantification of irinotecan and its active metabolite 7-ethyl-10-hydroxycamptothecin (SN38) in mouse plasma.

A simple, fast and reliable high-performance liquid chromatography-triple quadrupole mass spectrometry method (HPLC-MS/MS method) was developed, validated and used for the simultaneous quantification of irinotecan and 7-ethyl-10-hydroxycamptothecin (SN38) in heparinized mouse plasma. Camptothecin was used as the internal standard. A single-step protein precipitation without evaporation and reconstitution steps was adopted as sample processing method. Our bioanalytical method was validated in compliance with the guidelines from the European Medicines Agency. The lower limit of quantification for both irinotecan and SN38 was 5 ng/mL. The calibration curves for both analytes fitted to a 1/x(2) weighted linear regression model and ranged from 5 to 1000 ng/mL. The intra-run and inter-run precisions were within 8.6%, and the intra-run and inter-run accuracies were within 96.4-103.9%. Our validated bioanalytical method was successfully applied to the pharmacokinetic study in mice, in which 4 mg/kg irinotecan was intraperitoneally injected.

Development and validation of a microfluidic chip-based nano-liquid chromatography-triple quadrupole tandem mass spectrometry method for a sensitive and reliable quantification of 7-ethyl-10-hydroxycamptothecin (SN38) in mouse plasma.

There is an increasing need for more sensitive analytical methods in pharmacokinetic studies, for example, for phase 0 clinical trials. A novel HPLC Chip-triple quadrupole mass spectrometer method (HPLC Chip-MS/MS method) for the quantification of 7-ethyl-10-hydroxycamptothecin (SN38) was developed, validated, and employed to the pharmacokinetic analysis of SN38 in ICR mice. Protein precipitation with a ratio of plasma/acetonitrile of 1:10 was chosen as the sample processing method. The nano-electrospray inserted in the microfluidic chip operated in positive mode, and selected reaction monitoring was used for quantification. Our bioanalytical method met all essential validation parameters-selectivity, accuracy, precision, dilution integrity, calibration curve, matrix effect, recovery, and different stability tests (benchtop, freeze-thaw, autosampler stability). The calibration curves (weight 1/x (2)) were linear for the range 50-10,000 pg/mL. Clogging was not observed until the end of the lifetime of the microfluidic chip (350-400 injections), and carryover was practically eliminated through the introduction of a step gradient elution program. After intraperitoneal injection of 0.1 mg/kg irinotecan, SN38 concentration could be measured up to 6 h with accuracy and precision. Thus, we developed a new, very sensitive HPLC Chip-MS/MS method for the determination of plasma SN38 that has been validated in compliance with guidelines from different regulation authorities.

Synthesis, cytotoxicity and topoisomerase inhibition properties of multifarious aminoalkylated indeno[1,2-c]isoquinolin-5,11-diones.

A number of mono- or diaminoalkylated indeno[1,2-c]isoquinolin-5,11-diones analogs of 1 were synthesized and evaluated for their DNA binding affinities, topoisomerase inhibition properties and antiproliferative activities against human cancer cell lines (HL60). Impact of the side chain connected to the aromatic D ring and to the N6 lactam position on the biological profile will be discussed.

Indeno[1,2-c]isoquinolin-5,11-diones conjugated to amino acids: Synthesis, cytotoxicity, DNA interaction, and topoisomerase II inhibition properties.

Three series of indeno[1,2-c]isoquinolin-5,11-dione-amino acid conjugates were designed and synthesized. Amino acids were connected to the tetracycle through linkers with lengths of n=2 and 3 atoms using ester (series I), amide (series II), and secondary amine (series III) functions. DNA binding was evaluated by thermal denaturation and fluorescence measurements. Lysine and arginine substituted derivatives with n=3 provided the highest DNA binding. Arginine derivative 32 (n=2, series II) and glycine derivative 34 (n=2, series III) displayed high topoisomerase II inhibition. Incrementing the length of the N-6 side chain from two to three methylene units provided a significant increase in DNA affinity but a substantial loss in topoisomerase II inhibition. The most cytotoxic compounds toward HL60 leukemia cells were 19, 33, and 34 displaying micromolar IC(50) values. When tested with the topoisomerase II-mutated HL60/MX2 cell line, little variation of IC(50) values was found, suggesting that topoisomerase II might not be the main target of these compounds and that additional targets could be involved.