Rationale: Poly(amidoamine) PAMAM dendrimers are highly water soluble and are used as flexible scaffolding or nanocontainers to conjugate, complex or encapsulate therapeutic drugs to overcome intrinsically weak characteristics such as solubilization in aqueous medium. To provide a reliable method for the quantitation of PAMAM dendrimers in aqueous medium, we report here a validation study which was developed in a complex wastewater matrix to evaluate the matrix effect in the electrospray ionization (ESI) source.
Methods: PAMAM dendrimers (generations G0 to G3) were identified and quantitated in aqueous medium using liquid chromatography interfaced to a hybrid quadrupole/time-of-flight mass analyzer. This approach used the high resolving power of isotopic clusters and mass accuracy of the instrument, with especial attention to the tandem mass spectrometric (MS/MS) capabilities. The formation of multiply charged ions of PAMAM dendrimers in the ESI source and their later fragmentation allowed fragmentation paths to be determined and structural assignments to be made.
Results: The analytical strategy allowed dendrimer identification with a high degree of confidence obtained by accurate mass and high resolution with mass errors below 5 ppm and 10 ppm in MS and MS/MS modes. The parameters of validation in spiked matrix were: limits of quantification in the range of 0.12 to 1.25 μM depending on the generation, linearity (R >0.996), repeatability (R.S.D. <6.7%) and reproducibility (R.S.D. <10.8%).
Conclusions: Accurate mass measurement, elemental composition, and charge state assignment through the resolution of isotopic clusters of product and precursor ions, confers enhanced confidence on PAMAM dendrimer characterization. This selectivity provided high discriminating capacity of PAMAM dendrimers against matrix interferences. Because of the reliable and reproducible quantitation by LC/ESI-QTOF-MS, analysis of PAMAM dendrimers in an aqueous matrix is feasible.
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