Pulse gradient, large-volume injection, high-throughput ultra-performance liquid chromatographic/tandem mass spectrometry bioanalysis for measurement of plasma amrubicin and its metabolite amrubicinol.

In this communication, we report the development of a new ultra-performance liquid chromatographic/tandem mass spectrometry (UPLC-MS-MS) assay for measurement of amrubicin (an anthracycline anti-cancer agent) and its active metabolite, amrubicinol, in plasma. The enhanced electrospray ionization signal intensity of the analytes achieved by modifying the mobile phase with formic acid was associated with improvement in the lower limit of quantification. These favorable effects were electrolyte concentration-dependent. In order to maximize assay throughput, we used methanol protein precipitation to prepare the plasma samples, and simplified sample preparation by injecting 40 microL of the supernatant containing methanol at 87.5% (v/v) directly onto the UPLC column without any intermediary solvent evaporation step. The large-volume injection of highly organic supernatant sample increased matrix and elutropic effects, but these drawbacks were respectively overcome by using a 5mM formic acid-modified mobile phase and a new pulse gradient method. To our knowledge, this is the first report successfully using large-volume injection of strong organic samples with UPLC-MS-MS bioanalysis. The pulse gradient elution also resulted in band compression and enhanced the robustness of the chromatography. The promising new approach illustrated herein is extremely straightforward to optimize, and may be used for UPLC-MS-MS bioanalytical assay of other compounds.

'LC-electrolyte effects' improve the bioanalytical performance of liquid chromatography/tandem mass spectrometric assays in supporting pharmacokinetic study for drug discovery.

The development of rapid and sensitive bioanalytical methods in a short time frame with acceptable levels of precision and accuracy is imperative for successful drug discovery. We previously reported that the use of a mobile phase containing an extremely low concentration of ammonium formate or formic acid increased analyte electrospray ionization (ESI) response and controlled against matrix effects. We designated these favorable effects 'LC-electrolyte effects'. In order to support rapid pharmacokinetic (PK) studies for drug discovery, we applied LC-electrolyte effects to the development of generic procedures that can be used to quickly generate reliable PK data for compound candidates. We herein demonstrate our approach using four model tested compounds (Compd-A, -B, -C, and -D). The analytical methods involve generic protein precipitation for sample clean-up, followed by application of fast liquid chromatographic (LC) gradients and the subsequent use of electrospray ionization tandem mass spectrometry (ESI-MS/MS) for individual measurement of the tested compounds in 20-microL plasma samples. Good linearity over the concentration range of 1.6 or 8-25000 ng/mL (r(2) > 0.99), precision (RSD, 0.45-13.1%), and accuracy (91-112%) were achieved through the use of a low dose of formic acid (0.4 mM or 0.015 per thousand) in the methanol/water-based LC mobile phase. The analytical method was quite sensitive, providing a lower limit of quantification of 1.6 pg on-column except for Compd-C (8 pg), and showed negligible ion suppression caused by matrix components. Finally, the assay suitability was demonstrated in simulated discovery PK studies of the tested compounds with i.v./p.o. dosing of rats. This new assay approach has been adopted with good results in our laboratory for many recent discovery PK studies.

[Synthesis and antitumor activity of 20-O-linked camptothecin ester derivatives].

AIM: To improve the profile of 20 (S)-camptothecin, a series of 20-O-linked camptothecin phenoxyacetic acid ester derivatives have been designed. METHODS: These derivatives were synthesized by the method of acylation. Their chemical structures were confirmed with 1HNMR, IR, MS, and HRMS. The cytotoxicities of the compounds were tested by MTT assay. The in vivo antitumor activities of these esters were evaluated against mouse liver tumor H22 in mice. RESULTS: Twelve derivatives of camptothecin ester are new compounds. CONCLUSION: In vitro and in vivo antitumor activity has indicated that some derivatives appeared significantly more effective than topotecan in the H22 mouse liver tumoral model.