Bio-analytical method based on MALDI-MS analysis for the quantification of CIGB-300 anti-tumor peptide in human plasma

• A quantitative bioanalytical method based on MALDI-TOF MS for CIGB-300, in human plasma, was developed and fully validated.
• Acetylated CIGB-300 as internal standard allowed a suitable quantification.
• An easy sample cleanup with no need of chromatographic separation, assured no plasma interferences during MS analysis.
• Stability of CIGB-300 and its internal standard was thoroughly demonstrated in human plasma.
• Established calibration range allowed clinical samples analysis.

A fully validated bio-analytical method based on Matrix-Assisted-Laser-Desorption/Ionization-Time of Flight Mass Spectrometry was developed for quantitation in human plasma of the anti-tumor peptide CIGB-300. An analog of this peptide acetylated at the N-terminal, was used as internal standard for absolute quantitation. Acid treatment allowed efficient precipitation of plasma proteins as well as high recovery (approximately 80%) of the intact peptide. No other chromatographic step was required for sample processing before MALDI-MS analysis. Spectra were acquired in linear positive ion mode to ensure maximum sensitivity. The lower limit of quantitation was established at 0.5 μg/mL, which is equivalent to 160 fmol peptide. The calibration curve was linear from 0.5 to 7.5 μg/mL, with R2 > 0.98, and permitted quantitation of highly concentrated samples evaluated by dilution integrity testing. All parameters assessed for five validation batches met the FDA guidelines for industry. The method was successfully applied to analysis of clinical samples obtained in a phase I clinical trial following intravenous administration of CIGB-300 at a dose of 1.6 mg/kg body weight. With the exception of Cmax and AUC, pharmacokinetic parameters were similar for ELISA and MALDI-MS methods.

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