A Novel Approach to Internal Standardization in LC/MS/MS Analysis; Sensitive LC/MS/MS Analysis of Gentamicin

摘要

A novel approach to internal standardization in LC/MS/MS bioanalysis has been demonstrated. A deuterium labeled derivatization reagent was used to make an ideal internal standard for a complicated multi-component drug substance, Gentamicin, for which no satisfactory internal standard was available. Derivatization within the assay is required to make the drug substance analogous to the I.S. There are two critical characteristics for successful use of this unorthodox approach when the I.S. does not undergo derivatization within the assay. The steps of sample pre-treatment ahead of derivatization must be equally suitable for both derivatized and non-derivatized forms of the analyte. Secondly, the derivatization step itself must be robust and reliable and not susceptible to analyte conversion from the labeled derivative into the non-labeled derivative. Once the design of the method meets these requirements, all of the benefits of parallel extraction, chromatographic separation and LC/MS/MS ionization are realized just like would be with a synthetically labeled drug analogue. Though derivatizations are more labor intensive than would otherwise be required using true labeled compounds or analogues, the quality of the data depicted by this successful validation and the very sensitive LLOQ would not have otherwise been obtainable.